Methods of treating solid tumors with CCR2 antagonists

ABSTRACT

The present disclosure provides, inter alia, methods of treating a solid-tumor by administering an effective amount of a Chemokine Receptor 2 (CCR2) antagonist. Also provided herein are methods of reducing the number of macrophages in a solid tumor microenvironment, said method comprising administering effective amount of a Chemokine Receptor 2 (CCR2) antagonist. In an additional aspect, the current disclosure further provides methods of increasing the number CD8+ T cells in a solid tumor microenvironment, said method comprising administering effective amount of a Chemokine Receptor 2 (CCR2) antagonist. In some embodiments, the CCR2 antagonist has the formula I:

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C § 119(e)to U.S. Provisional Application Ser. No. 62/614,923 filed Jan. 8, 2018,the disclosure of which is incorporated herein by reference in itsentirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

NOT APPLICABLE

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

BACKGROUND

Tumor-associated macrophages (TAMs) are present in a large number intumor tissues which enhance the cancer-promoting inflammation [1-3].TAMs contribute to the immunosuppressive tumor microenvironment (TME) bysecreting a number of chemokines that are crucial to the recruitment ofimmunosuppressive cells. Furthermore, they produce angiogenetic factorssuch as VEGF, platelet-derived growth factor, and transforming growthfactor β to induce neovascularization. Moreover, PD-L1 (also known asB7H1) on macrophages confers TAMs with direct suppressive function byinducing antigen-specific tolerance in tumor-bearing hosts [3-5].

The abundance of macrophages in the TME and inverse correlation withsurvival has been frequently reported in malignancies, includingprostate, breast, colorectal, pancreas, and lymphomas [4, 6]. Highmacrophage density in tumors are associated with poor patient prognosisand treatment resistance, and has fueled cancer therapeutic strategiestargeting TAMs [7]. Of note, the presence of TAMs in human non-Hodgkin'slymphoma has been shown to not only correlate with patient's survivalbut also the responses to treatment [8]. Macrophage colony stimulatingfactor 1 receptor (CSF1R)-mediated signaling directs monocyte survivaland macrophage differentiation [9]. However, clinical trials applyingstrategies of CSF1R blockade are inconsistent in showing patientimprovement. The main reason for imperfect CSF1R inhibition may becaused by the dependency of the agent's ability to access malignantcells in the TME, potentially reducing the therapeutic effect of CSF1Rblockade[7, 10-12].

Blocking monocyte recruitment to tumors by targeting the CCL2-CCR2 axisprovides another promising strategy [13]. Neutralizing CCL2 antibodieshave been demonstrated to slow tumor progression in preclinical studies[14]. Clinical trials, however, showed limited clinical responses.Pharmacokinetic data revealed a rapid dissociation of the antibody andan undesired increase in serum CCL2 concentrations when targeting theCCL2/CCR2 axis in metastatic prostate cancer with a monoclonal CCL2antibody [15-18].

CCR2 antagonists have become attractive for targeting the CCL2-CCR2 axisin light of the limitations mentioned above [19, 20]. In a phase 1bstudy, CCR2 blockade by orally dosed small molecule CCR2 antagonist(PF-04136309) has demonstrated a reduction in TAM infiltration and anendogenous anti-tumor immune response in pancreatic ductaladenocarcinoma (PDAC)[21]. Overall, there is a scarcity of clinicaltrials with beneficial outcomes, and further studies are required toquantify the impact in different cancers. Mechanistically, how CCR2antagonists reshape the TME and how CCR2 antagonists activate anti-tumorimmunity yet remain to be described in preclinical tumor models withrespect to cellular immunomodulation. Additionally, optimized selectionand combination of standard chemotherapies for TAM targeting withradiotherapy or immunotherapy await development [22].

The present disclosure addresses these needs and provides relatedadvantages as well.

BRIEF SUMMARY

In one aspect, present disclosure provides methods of treating a solidtumor, said method comprising administering effective amount of aChemokine Receptor 2 (CCR2) antagonist.

In some embodiments, the tumor is a lymphoma. In some embodiments thelymphoma is cutaneous T cell lymphoma (CTCL).

In still another aspect, present disclosure provides methods of reducingthe number of macrophages in a solid tumor microenvironment, said methodcomprising administering effective amount of a Chemokine Receptor 2(CCR2) antagonist.

In yet another aspect, the present disclosure provides methods ofincreasing the number CD8+ T cells in a solid tumor microenvironment,said method comprising administering effective amount of a ChemokineReceptor 2 (CCR2) antagonist.

In some embodiments, the CCR2 receptor antagonist has the formula I

where each variable is described below.

In some embodiments, the CCR2 antagonist has the formula selected fromthe group consisting of

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula

or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C. Oral administration of Compound 1 inhibits tumor growth inthe MBL2/DNFB mouse model. (A) Scheme for the treatment regimen.Compound 1 is orally fed at 20 or 60 mg/kg daily for two weeks. Mice areeuthanized on day 15 for examining the ear tumors. Extra mice areeuthanized on day 3 or day 7 for determining earlier therapeuticresponses. (B) Examination on ear tumors in MBL2/DNFB mice which weretreated with two different doses of Compound 1 and vehicle for twoweeks. One representative ear from each group of 8 is shown. (C) Earthickness and ear weight are measured immediately after euthanasia onday 15 (*: p≤0.05, ***: p≤0.001).

FIG. 2A-B. Orally administration of Compound 1 is dose-dependentlyabsorptive and well tolerant in mice. (A) Mice were orally dosed forCompound 1 daily at a lower concentration (20 mg/kg per day) or a higherconcentration (60 mg/kg per day) for consecutive ten days. Plasmaconcentration of Compound 1 was detected by chemistry analyst 24 hoursafter the last dosing. (B) The same groups of mice in panel (A) wererecorded for body weight before the first and after the last oraladministration (n=4 per group). Statistical analysis is performed bytwo-way ANOVA in GraphPad PRISM (GraphPad Software, San Diego, Calif.).

FIG. 3A-C. Compound 1 specifically targets macrophages, but notneutrophils. (A, B) Macrophages, defined by the CD11b+/F4/80+ cellpopulations in flow analysis, were presented in either percentage orabsolute numbers in ear TME after only two daily doses of Compound 1(dosages are indicated in the figure, * p<0.05, ** p<0.01 vs vehiclecontrol). (C) Single cell suspension from the same tissues as in (A)were stained with antibodies for CD11b, F4/80, CCR2, Ly6G, and Ly6C.Cells gated on CD11b were further analyzed to differentiate thesubpopulations. Solid circles indicate the cells which are targeted byCompound 1. Dotted circles circles indicate the cells which are notblocked by Compound 1.

FIG. 4A-C. Enhanced inflammation is associated with CCR2 antagonism byCompound 1 in the tumor microenvironment. (A, B) Ear tissues werecollected from mice treated by Compound 1 (60 mg/kg) or vehicle on day7. Representative images for HE sections and IHC staining withanti-F4/80 were shown for the ear tissues from Compound 1 and vehicletreated group. (C) Ears from day 7 were also analyzed by flow cytometryto quantify the numbers of the two major myeloid subpopulations in TMEwith antibodies for CD11b, F4/80 and Ly6G.

FIG. 5A-C. Compound 1 treatment altered the expression of cytokines andbiomarkers produced from the TME. Quantitative RT-PCR was performed forthe ear tissues collected on day 7. Genes that are involved in cancerinflammation and immunity crosstalk are selectively detected.Comparative expression is performed between the Compound 1 and vehicletreated groups. (A) Shows immune stimulatory cytokines and cytotoxicactivation markers; (B) shows pro-inflammatory cytokines; and (C) showsneutrophil chemoattractants and biomarkers. Gene expression values arenormalized to endogenous expression of GAPDH (* p<0.05, ** p<0.01; n=3mice per group).

FIG. 6. Compound 1 treatment altered the expression of cytokines andbiomarkers produced from the TME. Quantitative RT-PCR was performed forthe ear tissues collected on day 7. Genes that are involved in immunesuppression and anti-inflammation are selectively detected. Comparativeexpression is performed between the Compound 1 and vehicle treatedgroups. Gene expression values are normalized to endogenous expressionof GAPDH (n=3 mice per group).

FIG. 7A-E. CD8 T cells are compulsory in CCR2 antagonist-mediatedanti-tumor immunity. (A) Tumor tissues were collected from miceeuthanized after two weeks of treatment. Groups are as indicated in thegraph. IHC staining with CD8a antibody was performed. The numbers of CD8positive T cells were counted by taking three random images of HPF (highpower field) in each section from all four mice per group. (B) Schemefor neutralizing CD8 T cells along with Compound 1 treatment.Neutralizing anti-CD8 or rat-IgG2a was administrated via intraperitonealinjection one day before MBL2 tumor inoculation followed by a seconddose after 7 days. Tumor formation was examined in two weeks after thetreatment with Compound 1 or vehicle. (C) Mice treated in experiment (B)were euthanized on day 3 for flow analysis of the cervical draininglymph nodes in order to determine the effect of CD8 depletion (threemice per group). (D) Mice were euthanized on day 15 after the two weeksof Compound 1/vehicle treatment with or without CD8 T cellneutralization. Ear thickness is measured for presenting tumor size. (E)Draining lymph nodes are also measured for the lymph node metastasis(n=4).

FIG. 8A-D. Compound 1 and anti-PD1 synergize the anti-tumor effect inMBL2 tumors. (A) qRT-PCR was performed to compare the expression of PD1and PD-L1 respectively in MBL2 tumors formed in ear skin compared to invitro cultured MBL2 cells. (B) Scheme for combining treatment withanti-PD1 with Compound 1 or vehicle control. (C) Spleens from the miceafter two weeks of treatment were processed for single cell suspensionfollowed by intracellular staining for flow analysis (Representativeflow graph from each group is shown). (D) Ear tumors were examined inmice euthanized after two weeks of treatment. Representative ear photofrom each group is shown (* p<0.05, n=8 mice per group). The dotted linein ear thickness bar graph indicates a borderline between a positive anda negative ability for tumor formation.

FIG. 9A-B. Anti-PD-L1 inhibits MBL2/DNFB tumor growth in mouse ears.Anti-PD-L1 (BioXcell, 150 ug per mouse, three times a week through IP)was administered starting from the same day of MBL2 tumor implantation.Mice were euthanized after two weeks of treatment. Ear tumor sizes andtumor weights were recorded. (A) Pictures are shown for two groups ofmice treated with either anti-PD-L1 or PBS control. (B) Ear thicknessand ear weight were measured (* p<0.05, ** p<0.01, n=4 mice per group)

DETAILED DESCRIPTION OF THE INVENTION I. General

The present disclosure is drawn, in part, to the surprising andunexpected finding that a CCR2 antagonist can be used to effectivelytreat a solid tumor and related lymphomas.

Cutaneous T cell lymphomas (CTCLs) are a heterogeneous group of T cellneoplasms that are primarily localized to skin, comprising the two mostcommon types, mycosis fungoides (MF) and Sézary syndrome (SS) [23].Evidence of skin inflammation is common in CTCLs [24, 25]. In lesionalskin of MF or SS, the numbers of CD163-positive macrophages areincreased and CC chemokine ligand 18 expression by macrophages promotesa T-helper (Th)2-dominant microenvironment by inducing chemotaxis of Th2cells. Such a tumor microenvironment is regarded as a determining factorto progressive clinical behavior of CTCL [26, 27]. By targeting TAMs inthe TME with a CCR2 antagonist, we provide alternative strategies forpatients at tumor-stage CTCL, where good therapeutic options are limit.

Prior reports have established a high grade T cell lymphoma model inmouse skin by injection of MBL2 T lymphoma cells in ear skin followed byapplication of 2,4-dinitro-1-fluorobenzene (DNFB)[28]. Tumor formationin this model is strictly dependent on the topical application of DNFB,which triggers an inflammatory skin response that promotes tumorformation. Herein, we demonstrate that Compound 1, a small molecule CCR2antagonist, depletes macrophages in the TME in the ear, leading tosignificantly more production of anti-tumor cytokines, such as IFN-γ.Administration of a CCR2 antagonist led to expansion of CD8 T cells andconsequently decreased the growth of implanted tumor cells. Thismechanism is supported by the observation that this anti-tumor effectcan be abrogated by simultaneously administering neutralizing CD8monoclonal antibody. Finally, we demonstrate that treatment efficacy ofthe CCR2 antagonist is increased by co-administration of anti-PD1antibody. Together, this report demonstrates that blocking therecruitment of TAMs into the TME may be an effective strategy fortreating T cell lymphoma and more generally, solid tumors.

II. Abbreviations and Definitions

CTCL (cutaneous T cell lymphoma); MF (mycosis fungoides); Tumormicroenvironment (TME); DNFB (2,4-dinitro-1-fluorobenzene); PD-1(programmed death ligand 1); qRT-PCR (Quantitative real-time PCR); TAMs(tumor-associated macrophages); IP (intraperitoneal); mAb (monoclonalantibody); IHC (immunohistochemistry).

The term “alkyl”, by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain hydrocarbonradical, having the number of carbon atoms designated (i.e. C₁₋₈ meansone to eight carbons). Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. The term “alkenyl” refers toan unsaturated alkyl group having one or more double bonds. Similarly,the term “alkynyl” refers to an unsaturated alkyl group having one ormore triple bonds. Examples of such unsaturated alkyl groups includevinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl).2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “cycloalkyl”refers to hydrocarbon rings having the indicated number of ring atoms(e.g., C₃₋₆cycloalkyl) and being fully saturated or having no more thanone double bond between ring vertices. “Cycloalkyl” is also meant torefer to bicyclic and polycyclic hydrocarbon rings such as, for example,bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. The term“heterocycloalkyl” refers to a cycloalkyl group that contain from one tofive heteroatoms selected from N, O, and S, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. The heterocycloalkyl may be a monocyclic, abicyclic or a polycylic ring system. Non limiting examples ofheterocycloalkyl groups include pyrrolidine, imidazolidine,pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin,dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine,thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide,piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone,tetrahydrofuran, tetrhydrothiophene, quinuclidine, and the like. Aheterocycloalkyl group can be attached to the remainder of the moleculethrough a ring carbon or a heteroatom. For terms such as cycloalkylalkyland heterocycloalkylalkyl, it is meant that a cycloalkyl or aheterocycloalkyl group is attached through an alkyl or alkylene linkerto the remainder of the molecule. For example, cyclobutylmethyl—is acyclobutyl ring that is attached to a methylene linker to the remainderof the molecule.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified by—CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group will have from 1to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingfour or fewer carbon atoms. Similarly, “alkenylene” and “alkynylene”refer to the unsaturated forms of “alkylene” having double or triplebonds, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S may beplaced at any interior position of the heteroalkyl group. The heteroatomSi may be placed at any position of the heteroalkyl group, including theposition at which the alkyl group is attached to the remainder of themolecule. Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the terms“heteroalkenyl” and “heteroalkynyl” by itself or in combination withanother term, means, unless otherwise stated, an alkenyl group oralkynyl group, respectively, that contains the stated number of carbonsand having from one to three heteroatoms selected from the groupconsisting of O, N, Si and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. The heteroatom(s) O, N and S may be placed at any interiorposition of the heteroalkyl group.

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent radical, saturated or unsaturated or polyunsaturated,derived from heteroalkyl, as exemplified by —CH₂—CH₂—S—CH₂CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—, —O—CH₂—CH═CH—, —CH₂—CH═C(H)CH₂—O—CH₂— and—S—CH₂—C═C—. For heteroalkylene groups, heteroatoms can also occupyeither or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy,alkyleneamino, alkylenediamino, and the like).

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively. Additionally, for dialkylaminogroups, the alkyl portions can be the same or different and can also becombined to form a 3-7 membered ring with the nitrogen atom to whicheach is attached. Accordingly, a group represented as —NR^(a)R^(b) ismeant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl andthe like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“C₁₋₄ haloalkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon group which can be a single ring ormultiple rings (up to three rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to five heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl groups include phenyl, naphthyl and biphenyl, whilenon-limiting examples of heteroaryl groups include pyridyl, pyridazinyl,pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl,quinazolinyl, cinnolinyl, phthalaziniyl, benzotriazinyl, purinyl,benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl,isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl,thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridines,benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl,isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl,triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl,thiazolyl, furyl, thienyl and the like. Substituents for each of theabove noted aryl and heteroaryl ring systems are selected from the groupof acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group thatis attached to the remainder of the molecule (e.g., benzyl, phenethyl,pyridylmethyl and the like).

The above terms (e.g., “alkyl,” “aryl” and “heteroaryl”), in someembodiments, will include both substituted and unsubstituted forms ofthe indicated radical. Preferred substituents for each type of radicalare provided below. For brevity, the terms aryl and heteroaryl willrefer to substituted or unsubstituted versions as provided below, whilethe term “alkyl” and related aliphatic radicals is meant to refer tounsubstituted version, unless indicated to be substituted.

Substituents for the alkyl radicals (including those groups oftenreferred to as alkylene, alkenyl, alkynyl and cycloalkyl) can be avariety of groups selected from: -halogen, —OR′, —NR′R″, —SR′,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR′S(O)₂R″, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″ and R′″ eachindependently refer to hydrogen, unsubstituted C₁₋₈ alkyl, unsubstitutedheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted C₁₋₈ alkyl, C₁₋₈ alkoxy or C₁₋₈ thioalkoxy groups, orunsubstituted aryl-C₁₋₄ alkyl groups. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 3-, 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant toinclude 1-pyrrolidinyl and 4-morpholinyl. The term “acyl” as used byitself or as part of another group refers to an alkyl radical whereintwo substitutents on the carbon that is closest to the point ofattachment for the radical is replaced with the substitutent ═O (e.g.,—C(O)CH₃, —C(O)CH₂CH₂OR′ and the like).

Similarly, substituents for the aryl and heteroaryl groups are variedand are generally selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′,—R′, —CN, —NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′,—NR″C(O)₂R′, —NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR'S(O)₂R″, —N₃,perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, C₁₋₈ alkyl, C₃₋₆ cycloalkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-C₁₋₄ alkyl, andunsubstituted aryloxy-C₁₋₄ alkyl. Other suitable substituents includeeach of the above aryl substituents attached to a ring atom by analkylene tether of from 1-4 carbon atoms.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A—(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—, —NH—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t), where s and t are independently integers of from0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—. Thesubstituent R′ in —NR’- and —S(O)₂NR′— is selected from hydrogen orunsubstituted C₁₋₆ alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

For the compounds provided herein, a bond that is drawn from asubstituent (typically an R group) to the center of an aromatic ring(e.g., benzene, pyridine, and the like) will be understood to refer to abond providing a connection at any of the available vertices of thearomatic ring. In some embodiments, the depiction will also includeconnection at a ring which is fused to the aromatic ring. For example, abond drawn to the center of the benzene portion of an indole, willindicate a bond to any available vertex of the six- or five-memberedring portions of the indole.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of salts derived frompharmaceutically-acceptable inorganic bases include aluminum, ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, zinc and the like. Salts derived frompharmaceutically-acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, malonic, benzoic, succinic, suberic,fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers, regioisomers and individual isomers (e.g., separateenantiomers) are all intended to be encompassed within the scope of thepresent invention. When compounds are provided herein with an identifiedstereochemistry (indicated as R or S, or with dashed or wedge bonddesignations), those compounds will be understood by one of skill in theart to be substantially free of other isomers (e.g., at least 80%, 90%,95%, 98%, 99%, and up to 100% free of the other isomer).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. Unnatural proportions of an isotope may bedefined as ranging from the amount found in nature to an amountconsisting of 100% of the atom in question. For example, the compoundsmay incorporate radioactive isotopes, such as for example tritium (³H),iodine-125 (¹²⁵I) or carbon-14 (¹⁴C), or non-radioactive isotopes, suchas deuterium (²H) or carbon-13 (¹³C). Such isotopic variations canprovide additional utilities to those described elsewhere within thisapplication. For instance, isotopic variants of the compounds of theinvention may find additional utility, including but not limited to, asdiagnostic and/or imaging reagents, or as cytotoxic/radiotoxictherapeutic agents. Additionally, isotopic variants of the compounds ofthe invention can have altered pharmacokinetic and pharmacodynamiccharacteristics which can contribute to enhanced safety, tolerability orefficacy during treatment. All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are intended to beencompassed within the scope of the present invention.

As used herein, the term “solid tumor” refers to a malignant neoplasm. Asolid tumors is generally localized mass of tissue; however, solidtumors are able to invade surrounding tissue and metastasize to new bodysides. Solid tumors may be benign (not cancer), or malignant (cancer).Different types of solid tumors are named for the type of cells thatform them. Examples of solid tumors are sarcomas, carcinomas, andlymphomas. The term “solid tumor” does not include leukemia. (cancers ofthe blood). “Sarcomas” are cancers arising from connective or supportingtissues such as bone or muscle. “Carcinomas” are cancers arising fromglandular cells and epithelial cells, which line body tissues.“Lymphomas” are cancers of the lymphoid organs such as the lymph nodes,spleen, and thymus. As these cells occur in most tissues of the body,lymphomas may develop in a wide variety of organs. Exemplary solidtumors include but are not limited to sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatocellular carcinoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioblastoma multiforme,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, cutaneous T celllymphoma (CTCL), melanoma, neuroblastoma, and retinoblastoma.

III. Detailed Description of Embodiments

A. Methods

In one aspect, the present disclosure provides methods of treating asolid tumor, said method comprising administering effective amount of aChemokine Receptor 2 (CCR2) antagonist.

In some embodiments the solid tumor is fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatocellular carcinoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioblastoma multiforme,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, cutaneous T celllymphoma (CTCL), melanoma, neuroblastoma, and retinoblastoma.

In some embodiments, the solid tumor is brain cancer, breast cancer,triple negative breast cancer, bladder cancer, bone cancer, colorectalcancer, lung cancer, kidney cancer, liver cancer, stomach cancer,prostate cancer, sarcoma, melanoma, carcinoma, or a lymphoma.

In some embodiments, the solid tumor is prostate cancer, breast cancer,colorectal cancer, pancreatic cancer, or a lymphoma.

In some embodiments the solid tumor is a lymphoma. In some embodiments,the lymphoma is cutaneous T cell lymphoma (CTCL). As described abovecutaneous T cell lymphomas (CTCLs) are a heterogeneous group of T cellneoplasms primarily localized to the skin.

CTCL is commonly broken into four separate stages (includingsub-stages). Early stage CTCL (Stage IA and IB) includes the skin beingcovered in red patches or plaques. The difference between Stage IA andStage IB is the amount of skin affected by red patches or plaques. AtStage IIA in additional to skin patches/plaques, the lymph nodes ofaffected individuals are enlarged, but the cancer has not spread to thenotes. Stage IIB is the stage where one or more tumors are found on theskin (i.e., “tumor-stage CTCL”), the lymph nodes may be enlarged, butcancer has not spread to the lymph nodes. In Stage III CTCL, nearly allof the skin is reddened including patches, plaques, and/or tumors, lymphnodes may be enlarged, but cancer has not spread to the lymph nodes. InStage IV, the cancer has spread to the lymph nodes or to other organs.

The present disclosure contemplates treating any of stages I-IV with themethods described herein. In some embodiments, subjects have early stageCTCL (i.e. Stage IA, IB, or IIA). In some embodiments, subjects withCTCL are in Stage IIB or a more advanced stage (i.e., the “tumor-stageCTCL). Thus, in some embodiments, the subject is diagnosed with StageIIB or a more advanced form of CTCL. In some embodiments, the subject isdiagnosed with Stage IB CTCL.

In some embodiments, the CTCL is a specific subtype of CTCL. In someembodiment, the CTCL is mycosis fungoides (MF). In some embodiments, theCTCL is Sézary syndrome (SS).

In a second aspect, the present disclosure provides methods of reducingthe number of macrophages in a solid tumor microenvironment, said methodcomprising administering effective amount of a Chemokine Receptor 2(CCR2) antagonist.

In a third aspect, the present disclosure provides methods of increasingthe number CD8+ T cells in a solid tumor microenvironment, said methodcomprising administering effective amount of a Chemokine Receptor 2(CCR2) antagonist.

B. CCR2 Antagonists

In some embodiments, the CCR2 antagonist is a small molecule antagonistof CCR2 having the formula (I):

or a pharmaceutically acceptable salt, hydrate, stereoisomer or rotamerthereof; wherein A is C(R⁵)(R⁶) or N(R⁵)

-   the subscripts m and n are each independently integers of from 0 to    2, and m+n is ≤3;-   R¹ is selected from the group consisting of aryl, aryl-C₁₋₄ alkyl,    heteroaryl and heteroaryl-C₁₋₄ alkyl, wherein the heteroaryl portion    has from 1-3 heteroatoms as ring members selected from N, O and S;    and wherein said aryl and heteroaryl groups or portions are    optionally substituted with from 1 to 5 R^(x) substituents;-   R² is selected from the group consisting of H, C₁₋₈ alkyl, C₃₋₈    cycloalkyl, C₃₋₈ cycloalkyl-C₁₋₄ alkyl, aryl, aryl-C₁₋₄ alkyl,    heteroaryl and heteroaryl-C₁₋₄ alkyl, wherein the heteroaryl portion    has from 1-3 heteroatoms as ring members selected from N, O and S;    and wherein said aryl and heteroaryl groups or portions are    optionally substituted with from 1 to 4 R^(x) substituents;-   or optionally, R¹ and R² are combined with the nitrogen atom to    which each is attached to form a 6- to 11-membered monocyclic or    fused bicyclic-heterocyclic or heteroaryl ring, wherein the —NR¹R²    is optionally further substituted with from 1 to 4 R^(x)    substituents;-   R³ is selected from the group consisting of H, C₁₋₈ alkyl, C₃₋₈    cycloalkyl and C₃₋₈ cycloalkyl-C₁₋₄ alkyl, each of which is    optionally substituted with from 1-3 R^(y) substituents;-   R⁴ is selected from the group consisting of H, C₁₋₈ alkyl optionally    substituted with 1 to 2 R^(y), and —CO₂H:-   R⁵ is selected from the group consisting of C₁₋₈ alkyl, C₁₋₈ alkoxy,    C₃₋₈ cycloalkyl, C₃₋₈ cycloalkyloxy, C₃₋₈ cycloalkyl-C₁₋₄ alkyl,    C₁₋₈ alkylamino, di-C₁₋₈ alkylamino, aryl, aryloxy, arylamino,    aryl-C₁₋₄ alkyl, heteroaryl, heteroaryloxy, heteroarylamino and    heteroaryl-C₁₋₄ alkyl, each of which is optionally substituted with    from 1 to 5 R^(z) substituents;-   R⁶ is selected from the group consisting of H, F, OH, C₁₋₈ alkyl and    C₁₋₈ alkoxy, wherein the C₁₋₈ alkyl and C₁₋₈ alkoxy groups are    optionally substituted with from 1 to 3 R^(z) substituents;-   or optionally, R⁵ and R⁶ are joined to form a spirocyclic 5- or    6-membered cycloalkyl ring which is optionally unsaturated, and has    a fused aryl group which is optionally substituted with from 1 to 4    R^(z) substituents;-   each R^(x) is independently selected from the group consisting of    -   halogen, —CN, —R^(c), —CO₂R^(a), —CONR^(a)R^(b), —C(O)R^(a),        —OC(O)NR^(a)R^(b), —NR^(b)C(O)R^(a), —NR^(b)C(O)₂R^(c),        —NR^(a)—C(O)NR^(a)R^(b), —NR^(a)C(O)NR^(a)R^(b), —NR^(a)R^(b),        —OR^(a), —O—X¹—OR^(a), —O—X¹—NR^(a)R^(b), —O—X¹—CO₂R^(a),        —O—X¹—CONR^(a)R^(b), —X¹—OR^(a), —X¹—NR^(a)R^(b), —X¹—CO₂R^(a),        —X¹—CONR^(a)R^(b), —SF₅, —S(O)₂NR^(a)R^(b), and 5- or 6-membered        aryl or heteroaryl, wherein each X¹ is a C₁₋₄ alkylene; each        R^(a) and R^(b) is independently selected from hydrogen, C₁₋₈        alkyl, and C₁₋₈ haloalkyl, or when attached to the same nitrogen        atom can be combined with the nitrogen atom to form a five or        six-membered ring having from 0 to 2 additional heteroatoms as        ring members selected from N, O or S, and optionally substituted        with oxo; each R^(c) is independently selected from the group        consisting of C₁₋₈ alkyl, C₁₋₈ haloalkyl and C₃₋₆ cycloalkyl,        and optionally when two R^(X) substituents are on adjacent        atoms, are combined to form a fused five or six-membered        carbocyclic ring, and wherein the aryl or heteroaryl groups are        optionally substituted with 1-3 members selected from halogen,        hydroxyl, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkyl, and C₁₋₄        haloalkoxy;-   each R^(y) is independently selected from the group consisting of    -   halogen, —CN, —R^(f), —CO₂R^(d), —CONR^(d)R^(e), —C(O)R^(d),        —OC(O)NR^(d)R^(e), —NR^(e)C(O)R^(d), —NR^(e)C(O)₂R^(f),        —NR^(d)C(O)NR^(d)R^(e), —NR^(d)C(O)NR^(d)R^(e), —NR^(d)R^(e),        —OR^(d), and —S(O)₂NR^(d)R^(e); wherein each R^(d) and R^(e) is        independently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈        haloalkyl, or when attached to the same nitrogen atom can be        combined with the nitrogen atom to form a five or six-membered        ring having from 0 to 2 additional heteroatoms as ring members        selected from N, O or S; each R^(f) is independently selected        from the group consisting of C₁₋₈ alkyl, C₁₋₈ haloalkyl and C₃₋₆        cycloalkyl;-   each R^(z) is independently selected from the group consisting of    halogen, —CN, —R^(i), —CO₂R^(g), —CONR^(g)R^(h), —C(O)R^(g),    —OC(O)NR^(g)R^(h), —NR^(h)C(O)R^(g), —NR^(h)C(O)₂R^(i),    —NR^(g)C(O)NR^(g)R^(h), —NR^(g)R^(h), —OR^(g), —S(O)₂NR^(g)R^(h),    —X¹—R^(j), —X¹—NR^(g)R^(h), —X¹—CONR^(g)R^(h), —X¹—NR^(h)C(O)R^(g),    —NHR^(j), —NHCH₂R^(j), and tetrazole; wherein each R^(g) and R^(h)    is independently selected from hydrogen, C₁₋₈ alkyl, C₃₋₆ cycloalkyl    and C₁₋₈ haloalkyl, or when attached to the same nitrogen atom can    be combined with the nitrogen atom to form a five or six-membered    ring having from 0 to 2 additional heteroatoms as ring members    selected from N, O or S and is optionally substituted with one or    two oxo; each R^(i) is independently selected from the group    consisting of C₁₋₈ alkyl, C₁₋₈ haloalkyl and C₃₋₆ cycloalkyl; and    each R is selected from the group consisting of C₃₋₆ cycloalkyl,    pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, and    tetrahydropyranyl.

It shall be understood that when R¹ and R² are combined with thenitrogen atom to which each is attached to form a 6- to 11-memberedmonocyclic or fused bicyclic-heterocyclic ring, the 6- to 11-memberedmonocyclic or fused bicyclic-heterocyclic ring encompasses monocyclicheterocyclic rings fused with an aryl or a heteroaryl ring.

In formula I, the substituent R³ is, in one embodiment, selected fromthe group consisting of H, methyl, ethyl, propyl, isopropyl, buty,isobutyl, sec-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl andcyclobutylmethyl.

In the descriptions herein, one of skill in the art will understand thatthe wavy line intersecting a bond is meant to identify the point ofattachment of a given substituent or group to the remainder of themolecule.

As noted above, the subscripts m and n are each integers selected from0, 1 and 2, and m+n is ≤3. When the subscript is 0, one of skill in theare will understand that a cyclic structure with ring vertex A isintended, but that adjacent ring vertices on either side of theparentheses are joined by a bond. Accordingly, the present inventionincludes the structures wherein the ring having A as a vertex is meantto include:

In one selected group of embodiments, m and n are both 0. In anotherselected group of embodiments, m and n are both 1. In yet another groupof selected embodiments, m is 1 and n is 0. In still another group ofembodiments, m is 1 and n is 2.

In still other selected embodiments, the ring having vertex A isrepresented by a formula selected from:

In one subgroup of embodiments, the compounds of formula (I) arerepresented by:

Within formula (Ia), a number of selected embodiments are provided asformulae Ia1, Ia2, Ia3, Ia4 and Ia5.

In each of formulae Ia, Ia1, Ia2, Ia3, Ia4 and Ia5, the notedsubstituents (R¹ through R⁶, R^(x) and R^(z)) and subscripts m and nhave the meanings provided above with respect to formula I. Thesubscripts, p and q, have the following meanings: for Ia1, Ia4 and Ia5,the subscript q is an integer of from 0 to 5; for Ia2 and Ia4, thesubscript p is an integer of from 0 to 4; and for Ia3 and Ia5, thesubscript p is an integer of from 0 to 5.

In still other selected embodiments, the compounds provided herein arerepresented by formulae selected from:

wherein each compound is substantially free of other stereoisomers, andwherein the noted substituents (R¹ through R⁶, R^(x) and R^(z)) andsubscripts m and n have the meanings provided above with respect toformula I. The subscripts, p and q, have the following meanings: forIa1′, Ia4′ and Ia5′, the subscript q is an integer of from 0 to 5; forIa2′ and Ia4′, the subscript p is an integer of from 0 to 4; and forIa3′ and Ia5′, the subscript p is an integer of from 0 to 5.

In another group of embodiments of formula I, A is C(R⁵)(R⁶), wherein R⁵and R⁶ are combined to form a ring. Selected embodiments are provided asfollows:

In each of formulae Ib, Ib1 and Ib2, the noted substituents (R¹ throughR⁶, R^(x) and R^(z)) and subscripts m and n have the meanings providedabove with respect to formula I. The subscripts, p and q, have thefollowing meanings: for Ib, Ib1 and Ib2, the subscript q is an integerof from 0 to 5; for Ib1, the subscript p is an integer of from 0 to 4;and for Ib2, the subscript p is an integer of from 0 to 5.

In another group of embodiments of formula I, A is NR⁵ (see formula Ic).Selected embodiments are provided as follows:

In each of formulae Ic, Ic1, Ic2, Ic3, Ic4 and Ic5, the notedsubstituents (R¹ through R⁶, R^(x) and R^(z)) and subscripts m and nhave the meanings provided above with respect to formula I. Thesubscripts, p and q, have the following meanings: for Ic1, Ic4 and Ic5,the subscript q is an integer of from 0 to 5; for Ic2 and Ic4, thesubscript p is an integer of from 0 to 4; and for Ic3 and Ic5, thesubscript p is an integer of from 0 to 5.

In still other selected embodiments, the compounds provided herein arerepresented by formulae selected from:

wherein each compound is substantially free of other stereoisomers, andwherein the noted substituents (R¹ through R⁶, R^(x) and R^(z)) andsubscripts m and n have the meanings provided above with respect toformula I. The subscripts, p and q, have the following meanings: forIc1′, Ic4′ and Ic5′, the subscript q is an integer of from 0 to 5; forIc2′ and Ic4′, the subscript p is an integer of from 0 to 4; and forIc3′ and Ic5′, the subscript p is an integer of from 0 to 5.

Other selected embodiments, compounds are provided in each of I, Ia,Ia1, Ia1′, Ib, Ic, Ic1 and Ic1′, described above, wherein —N(R¹)(R²) isselected from:

Still other selected embodiments, are provided in each of I, Ia, Ia1,Ia1′, Ib, Ic, Ic1 and Ic1′, described above, wherein —N(R¹)(R²) isselected from:

Yet other selected embodiments, are provided in each of I, Ia, Ia1,Ia1′, Ib, Ic, Ic1 and Ic1′, described above, wherein —N(R¹)(R²) isselected from:

In some embodiments, compounds of formulae I, Ia, Ia2, Ia3, Ia2′ andIa3′, are provided wherein A is C(R⁵)(R⁶), or is shown in the formula asC(R⁵)(R⁶), wherein R⁵ is selected from aryl, aryloxy, arylamino,aryl-C₁₋₄ alkyl, heteroaryl, heteroaryloxy, heteroarylamino andheteroaryl-C₁₋₄ alkyl, wherein the aryl or heteroaryl groups or portionsare selected from:

In certain selected embodiments, compounds of formulae I, Ia, Ia2, Ia3,Ia2′ and Ia3′, are provided wherein A is C(R⁵)(R⁶), or is shown in theformula as C(R⁵)(R⁶), wherein R⁵ is selected from aryl, aryloxy,arylamino and aryl-C₁₋₄ alkyl, wherein the aryl group or portion isselected from:

In still other selected embodiments, compounds of formulae I, Ia, Ia2,Ia3, Ia2′ and Ia3′, are provided wherein A is C(R⁵)(R⁶), or is shown inthe formula as C(R⁵)(R⁶), wherein R⁵ is selected from heteroaryl,heteroaryloxy, heteroarylamino and heteroaryl-C₁₋₄ alkyl, wherein theheteroaryl group or portion is selected from:

In some embodiments, compounds of formulae I, Ic, Ic2, Ic3, Ic2′ andIc3′, are provided wherein A is N(R⁵), or is shown in the formula asN(R′), wherein R¹ is selected from aryl, aryl-C₁₋₄ alkyl, heteroaryl andheteroaryl-C₁₋₄ alkyl, wherein the aryl or heteroaryl groups or portionsare selected from Group 1 above. In certain selected embodiments,compounds of formulae I, Ic, Ic2, Ic3, Ic2′ and Ic3′, are providedwherein A is N(R⁵), or is shown in the formula as N(R⁵), wherein R⁵ isselected from aryl and aryl-C₁₋₄ alkyl, wherein the aryl group orportion is selected from Subgroup 1a, above. In still other selectedembodiments, compounds of formulae I, Ic, Ic2, Ic3, Ic2′ and Ic3′, areprovided wherein A is N(R⁵), or is shown in the formula as N(R⁵),wherein R⁵ is selected from heteroaryl and heteroaryl-C₁₋₄ alkyl,wherein the heteroaryl group or portion is selected from Subgroup 1b,above.

In some embodiments, the CCR2 antagonist has the formula selected fromthe group consisting of

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula of Compound 1

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula of Compound 2

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula of Compound 3

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist is selected from the compoundsor pharmaceutical compositions disclosed in US2016/0340356, stemmingfrom application Ser. No. 15/158,713, filed on May 19, 2016 byChemoCentryx, the content of which is incorporated herein for allpurposes.

In some embodiments, the CCR2 chemokine receptor antagonist is selectedfrom the group consisting of AZ889, AZD2423, INCB-8761, MK-0812,BMS-813160, INCB-003284, PF-04634817, BMS-741672, Cenicriviroc, CCX-140.

C. Methods of Administration

The term “therapeutically effective amount” means the amount of thesubject compound that will elicit the biological or medical response ofa cell, tissue, system, or animal, such as a human, that is being soughtby the researcher, veterinarian, medical doctor or other treatmentprovider.

In general, treatment methods provided herein comprise administering toa patient an effective amount of a compound one or more compoundsprovided herein. In a preferred embodiment, the compound(s) of theinvention are preferably administered to a patient (e.g., a human)orally or topically. Treatment regimens may vary depending on thecompound used and the particular condition to be treated; for treatmentof most disorders, a frequency of administration of 4 times daily orless is preferred. In general, a dosage regimen of 2 times daily is morepreferred, with once a day dosing particularly preferred. It will beunderstood, however, that the specific dose level and treatment regimenfor any particular patient will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination (i.e., other drugsbeing administered to the patient) and the severity of the particulardisease undergoing therapy, as well as the judgment of the prescribingmedical practitioner. In general, the use of the minimum dose sufficientto provide effective therapy is preferred. Patients may generally bemonitored for therapeutic effectiveness using medical or veterinarycriteria suitable for the condition being treated or prevented.

Depending on the disease to be treated and the subject's condition, thecompounds and compositions of the present invention may be administeredby oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous,ICV, intracisternal injection or infusion, subcutaneous injection, orimplant), inhalation, nasal, vaginal, rectal, sublingual, or topicalroutes of administration and may be formulated, alone or together, insuitable dosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants and vehicles appropriatefor each rouse of administration. The present invention alsocontemplates administration of the compounds and compositions of thepresent invention in a depot formulation.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment orpreventions of conditions involving pathogenic CCR2 activity (about 0.5mg to about 7 g per human patient per day). The amount of activeingredient that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. Dosage unit forms will generallycontain between from about 1 mg to about 500 mg of an active ingredient.For compounds administered orally, transdermally, intravaneously, orsubcutaneously, it is preferred that sufficient amount of the compoundbe administered to achieve a serum concentration of 5 ng(nanograms)/mL-10 μg (micrograms)/mL serum, more preferably sufficientcompound to achieve a serum concentration of 20 ng-1 μg/ml serum shouldbe administered, most preferably sufficient compound to achieve a serumconcentration of 50 ng/ml-200 ng/ml serum should be administered. Fordirect injection into the synovium (for the treatment of arthritis)sufficient compounds should be administered to achieve a localconcentration of approximately 1 micromolar.

Frequency of dosage may also vary depending on the compound used and theparticular disease treated. However, for treatment of most disorders, adosage regimen of 4 times daily, three times daily, or less ispreferred, with a dosage regimen of once daily or 2 times daily beingparticularly preferred. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diet, time ofadministration, route of administration, and rate of excretion, drugcombination (i.e., other drugs being administered to the patient), theseverity of the particular disease undergoing therapy, and otherfactors, including the judgment of the prescribing medical practitioner.

In some embodiments, the treatment or prevention of conditions whichrequire CCR2 receptor modulation, an appropriate dosage level willgenerally be about 0.001 to 100 mg per kg patient body weight per daywhich can be administered in single or multiple doses. Preferably, thedosage level will be about 0.01 to about 25 mg/kg per day; morepreferably about 0.05 to about 10 mg/kg per day. A suitable dosage levelmay be about 0.01 to 25 mg/kg per day, about 0.05 to 10 mg/kg per day,or about 0.1 to 5 mg/kg per day. Within this range the dosage may be0.005 to 0.05, 0.05 to 0.5, 0.5 to 5.0, or 5.0 to 50 mg/kg per day. Fororal administration, the compositions are preferably provided in theform of tablets containing 1.0 to 1000 milligrams of the activeingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0,100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0,900.0, and 1000.0 milligrams of the active ingredient for thesymptomatic adjustment of the dosage to the patient to be treated. Thecompounds may be administered on a regimen of 1 to 4 times per day,preferably once or twice per day.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, hereditary characteristics, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and the hostundergoing therapy.

D. Combination Therapy

In treating, preventing, ameliorating, controlling or reducing solidtumor growth and metastases, the compounds of the present invention maybe used in conjunction with the following: (1) cancer vaccinationstrategies, (2) immune-checkpoint modulators such as antagonisticantibodies against immune-checkpoint inhibitors (anti-PD1, anti-PD-L1,anti-CTLA4, anti-Tim3, anti-VISTA, anti-KIR) or agonistic antibodiesagainst immune-accelators (anti-Lag3, anti-OX40, anti-ICOS, anti-4-1BB,(3) blocking or depleting antibodies against cell surface proteinscommonly up-regulated in transformed cells (CEACAM1, Syndecan-2, GRP78),(4) anti-angiogenic therapies (anti-VEGF, anti-VEGFR, VEGFR smallmolecule inhibitors), (5) anti-lymphangiogenesis (blocking antibodies orinhibitors against VEGF, FDF2, PDGF as well as its respectivereceptors), (6) standard chemotherapeutic therapies (Gemcitabine,Paclitaxel, FOLFORINOX), (7) irradiation therapy, (8) other chemokineantagonists (CCR1, CCR4, CCR6, CXCR4, CXCR2, CXCR7 small moleculeinhibitors, blocking antibodies, or depleting antibodies), (9) depletingantibodies against chemokines that activate the aforementioned chemokinereceptors, (10) inhibitors targeting common somatic mutations in cancersuch as those specifically targeting the following genes (BRAF, KRAS,NRAS, EGFR, CTNNB1, NOTCH1, PIK3CA, PTEN, APC, FLT3, IDH1, IDH2, KIT,TP53, JAK2). Combination therapy is also contemplated in methods ofincreasing the number CD8+ T cells in a solid tumor microenvironment andmethods of reducing the number of macrophages in a solid tumormicroenvironment.

In some embodiments, the compounds of the present invention may be usedin conjunction with an anti-inflammatory or analgesic agent such as anopiate agonist, a lipoxygenase inhibitor, such as an inhibitor of5-lipoxygenase, a cyclooxygenase inhibitor, such as a cyclooxygenase-2inhibitor, an interleukin inhibitor, such as an interleukin-1 inhibitor,an NMDA antagonist, an inhibitor of nitric oxide or an inhibitor of thesynthesis of nitric oxide, a non-steroidal antiinflammatory agent, or acytokine-suppressing anti-inflammatory agent, for example with acompound such as acetaminophen, aspirin, codeine, biological TNFsequestrants, fentanyl, ibuprofen, indomethacin, ketorolac, morphine,naproxen, phenacetin, piroxicam, a steroidal analgesic, sufentanyl,sunlindac, tenidap, and the like.

In some embodiments, the immune checkpoint inhibitor is a PD-1 and/orPD-L1 inhibitor. In some embodiments, a PD-L1 inhibitor can bedurvalumab or atezolizumab or avelumab or BMS-936559 (MDX-1105) orALN-PDL or TSR-042 or KD-033 or CA-170 or CA-327 or STI-1014 orMEDI-0680 or KY-1003. Durvalumab (MEDI4736) is a human monoclonalantibody directed against PD-L1. Atrexolizumab (MPDL3280A) is a fullyhumanized, engineered IgG1 monoclonal antibody against PD-L1. Avelumab(MSB0010718C) is a fully humanized, engineered IgG1 monoclonal antibodyagainst PD-L1. BMS-936559 (MDX-1105) is a fully human IgG4 monoclonalantibody against PD-L1. ALN-PDL is an inhibitory RNA (RNAi) targetingPD-L1. TSR-042 refers to an engineered chimeric antibody that isdirected against the PD-1/PD-L1 pathway. KD-033 refers to a bifunctionalanti-PD-L1/IL-15 fusion protein wherein the anti-PD-L1 antibody islinked at its tail to the cytokine IL-15 by the sushi domain of theIL-15 receptor. CA-170 refers to a small molecule antagonist of PD-L1and VISTA. STI-1014 refers to an anti-PD-L1 antibody. KY-1003 is amonoclonal antibody against PD-L1. CA-327 refers to a small moleculeantagonist of PD-L1 and TIM3.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is selected fromthe group consisting of durvalumab, atezolizumab, pembrolizumab,nivolumab, AP-106, AP-105, MSB-2311, CBT-501, avelumab, AK-105, IO-102,IO-103, PDR-001, CX-072, SHR-1316, JTX-4014, GNS-1480, recombinanthumanized anti-PD1 mAb (Shanghai Junshi Biosciences), REGN-2810,pelareorep, SHR-1210, PD1/PDL1 inhibitor vaccine (THERAVECTYS),BGB-A317, recombinant humanized anti-PD-1 mAb (Bio-Thera Solutions),Probody targeting PD-1 (CytomX), XmAb-20717, FS-118, PSI-001, SN-PDL01,SN-PD07, PD-1 modified TILs (Sangamo Therapeutics), PRS-332, FPT-155,jienuo mAb (Genor Biopharma), TSR-042, REGN-1979, REGN-2810,resminostat, FAZ-053, PD-1/CTLA-4 bispecific antibody (MacroGenics),MGA-012, MGD-013, M-7824, PD-1 based bispecific antibody (Beijing HanmiPharmaceutical), AK-112, AK-106, AK-104, AK-103, BI-754091, ENUM-244C8,MCLA-145, MCLA-134, anti-PD1 oncolytic monoclonal antibody (TransgeneSA), AGEN-2034, IBI-308, WBP-3155, JNJ-63723283, MEDI-0680, SSI-361,CBT-502, anti-PD-1 bispecific antibody, dual targeting anti-PD-1/LAG-3mAbs (TESARO), dual targeting anti-PD-1/TIM-3 mAbs (TESARO),PF-06801591, LY-3300054, BCD-100, STI-1110, pembrolizumab biosimilar,nivolumab biosimilar, PD-L1-TGF-beta therapy, KY-1003, STI-1014,GLS-010, AM-0001, GX-P2, KD-033, PD-L/BCMA bispecific antibody (ImmunePharmaceuticals), PD-1/Ox40 targeting bispecific antibody (ImmunePharmaceuticals), BMS-936559, anti-PD-1/VEGF-A DARPins (MolecularPartners), mDX-400, ALN-PDL, PD-1 inhibitor peptide (Aurigene), siRNAloaded dendritic cell vaccine (Alnylam Pharmaceuticals), GB-226, PD-L1targeting CAR-TNK-based immunotherapy (TNK Therapeutics/NantKwest),INSIX RA, INDUS-903, AMP-224, anti-CTLA-4/anti-PD-1 bispecific humanizedantibody (Akeso Biopharma), B7-H1 vaccine (State Key Laboratory ofCancer Biology/Fourth Military Medical University), and GX-D1.

In some embodiments, a PD-1 inhibitor can be pembrolizumab or nivolumabor IBI-308 or mDX-400 or BGB-108 or MEDI-0680 or SHR-1210 or PF-06801591or PDR-001 or GB-226 or STI-1110. Nivolumab (also known as OPDIVO™,MDX-1106, BMS-936558, and ONO-4538) is a human IgG4 monoclonal antibodyagainst PD-1. Pembrolizumab (also known as KEYTRUDA®, lambrolizumab, andMK-34) is a humanized IgG4 kappa isotype monoclonal antibody againstPD-1. IBI-308 refers to a monoclonal antibody directed to PD-1. mDX-400refers to a mouse antibody against PD-1. BGB-108 is a humanizedmonoclonal antibody against PD-1. MEDI-0680 (AMP-514) is a humanizedIgG4 monoclonal antibody against PD-1. SHR-1210 refers to a monoclonalantibody against PD-1. PF-06801591 is a monoclonal antibody againstPD-1. PDR-001 refers to a monoclonal antibody against PD-1. GB-226refers to a monoclonal antibody against PD-1. STI-1110 refers to amonoclonal antibody against PD-1.

In some embodiments, the PD-1 inhibitor is RPM1-14.

In some embodiments, the PD-1 inhibitor is an antibody selected fromNivolumab, Pembrolizumab, and Pidilizumab.

The anti-PD-1 antibodies, and antibody fragments described hereinencompass proteins having amino acid sequences that vary from those ofthe described antibodies, but that retain the ability to bind PD-1.

In some embodiments, the anti-PD-1 antibodies include bispecificantibodies and antibody-like therapeutic proteins including DARTs®,DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives, and the like thatbind to PD-1.

The anti-PD-L1 antibodies and antibody fragments described hereinencompass proteins having amino acid sequences that vary from those ofthe described antibodies, but that retain the ability to bind PD-L1.Such variant antibodies and fragments thereof can comprise one or moreadditions, deletions, or substitutions of amino acids when compared tothe parent sequence, but exhibit biological activity that is essentiallyequivalent or essentially bioequivalent to that of the describedantibodies.

In some embodiments, the anti-PD-L1 antibodies include bispecificantibodies and antibody-like therapeutic proteins including DARTs®,DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives, and the like thatbind to PD-L1.

Non-limiting examples of additional PD-1/PD-L1 pathway inhibitors aredescribed in, e.g., Chen and Han, Jour Clin Invest, 2015,125(9):3384-3391, U.S. Pat. Nos. 8,168,757; 8,354,509; 8,552,154;8,741,295; and 9,212,224; U.S. Patent App. Publ. Nos. 2014/0341917;2015/0203580 and 2015/0320859; International Patent App. Publ. No.WO2015/026634.

In some embodiments, the immune checkpoint inhibitor is a CTLA-4inhibitor. A number of CTLA-4 inhibitors are known in the art. In someembodiments, the CTLA-4 inhibitor is an antibody. In some embodimentsthe CTLA-4 inhibitor antibody is selected from Ipilimumab, Tremelimumab,AGEN1884, and AGEN2041. In some embodiments, the CTLA-4 inhibitorantibody is Ipilimumab. In some embodiments, the CTLA-4 inhibitorantibody is Tremelimumab. In some embodiments, the CTLA-4 inhibitorantibody is AGEN1884. In some embodiments, the CTLA-4 inhibitor antibodyis AGEN2041.

A biological product, e.g., an antibody or a fragment thereof, isconsidered a biosimilar if, for example, the biological product ishighly similar to an already FDA-approved biological product, known asthe reference product. A biosimilar has no clinically meaningfuldifferences in terms of safety and effectiveness from the referenceproduct. A biosimilar can also have the same mechanism of action, routeof administration, dosage form, and strength as its reference product.

Two biological products, e.g., antibodies or fragments thereof, areconsidered bioequivalent if, for example, they are pharmaceuticalequivalents or pharmaceutical alternatives whose rate and extent ofabsorption do not show a significant difference when administered at thesame molar dose under similar experimental conditions, either singledose or multiple doses. Some antibodies will be considered equivalentsor pharmaceutical alternatives if they are equivalent in the extent oftheir absorption but not in their rate of absorption and yet may beconsidered bioequivalent because such differences in the rate ofabsorption are intentional and are reflected in the labeling, are notessential to the attainment of effective body drug concentrations on,e.g., chronic use, and are considered medically insignificant for theparticular drug product studied.

In some embodiments, two biological products (e.g., two antibodies orfragments thereof) are bioequivalent if there are no clinicallymeaningful differences in their safety, purity, or potency.

In other embodiments, two biological products (e.g., two antibodies orfragments thereof) are bioequivalent if a patient can be switched one ormore times between the reference product and the biological productwithout an expected increase in the risk of adverse effects, including aclinically significant change in immunogenicity, or diminishedeffectiveness, as compared to continued therapy without such switching.

In yet other embodiments, two biological products (e.g., two antibodiesor fragments thereof) are bioequivalent if they both act by a commonmechanism of action for the condition of use, to the extent that suchmechanisms are known.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Biobetter variants of the antibodies described herein may be based on anexisting reference antibody specific for an target antigen, e.g., PD-1or PD-L1, which has undergone changes such that, for example, it has ahigher binding affinity to its target antigen and/or binds to adifferent epitope than the reference antibody, or has more desirabletherapeutic efficacy, expression and/or biophysical characteristics.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is a small moleculePD-1/PD-L1 inhibitor of having the formula:

In some embodiments, the PD-1 and/or PD-L1 inhibitor is a small moleculePD-1/PD-L1 inhibitor having the formula (II)

or a pharmaceutically acceptable salt thereof; wherein:

-   R¹ is selected from the group consisting of halogen, C₅₋₈    cycloalkyl, C₆₋₁₀ aryl and thienyl, wherein the C₆₋₁₀ aryl and    thienyl are optionally substituted with 1 to 5 R^(x) substituents;-   each R^(x) is independently selected from the group consisting of    halogen, —CN, —R^(c), —CO₂R^(a), —CONR^(a)R^(b), —C(O)R^(a),    —OC(O)NR^(a)R^(b), —NR^(b)C(O)R^(a), —NR^(b)C(O)₂R^(c),    —NR^(a)—C(O)NR^(a)R^(b), —NR^(a)R^(b), —OR^(a), —O—X¹—OR^(a),    —O—X¹—CO₂R^(a), —O—X¹—CONR^(a)R^(b), —X¹—OR^(a), —X¹—NR^(a)R^(b),    —X¹—CO₂R^(a), —X¹—CONR^(a)R^(b), —SF₅, and —S(O)₂NR^(a)R^(b),    wherein each X¹ is a C₁₋₄ alkylene; each R^(a) and R^(b) is    independently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈    haloalkyl, or when attached to the same nitrogen atom can be    combined with the nitrogen atom to form a five or six-membered ring    having from 0 to 2 additional heteroatoms as ring members selected    from N, O or S, wherein the five or six-membered ring is optionally    substituted with oxo; each R is independently selected from the    group consisting of C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl and C₁₋₈    haloalkyl; and optionally when two R^(x) substituents are on    adjacent atoms, they are combined to form a fused five, six or    seven-membered carbocyclic or heterocyclic ring optionally    substituted with from 1 to 3 substituents independently selected    from halo, oxo, C₁₋₈ haloalkyl and C₁₋₈ alkyl;-   each R^(2a), R^(2b) and R^(2c) is independently selected from the    group consisting of H, halogen, —CN, —R^(d), —CO₂R^(e),    —CONR^(e)R^(f), —C(O)R^(e), —OC(O)NR^(e)R^(f), —NR^(f)C(O)R^(e),    —NR^(f)C(O)₂R^(d), —NR^(e)—C(O)NR^(e)R^(f), —NR^(e)R^(f), —OR^(e),    —O—X²—OR^(e), —O—X²—NR^(e)R^(f), —O—X²—CO₂R^(e),    —O—X²—CONR^(e)R^(f), —X²—OR^(e), —X²—NR^(e)R^(f), —X²—CO₂R^(e),    —X²—CONR^(e)R^(f), —SF₅, —S(O)₂NR^(e)R^(f), C₆₋₁₀ aryl and C₅₋₁₀    heteroaryl, wherein each X² is a C₁₋₄ alkylene; each R^(e) and R^(f)    is independently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈    haloalkyl, or when attached to the same nitrogen atom can be    combined with the nitrogen atom to form a five or six-membered ring    having from 0 to 2 additional heteroatoms as ring members selected    from N, O and S, and optionally substituted with oxo; each R^(d) is    independently selected from the group consisting of C₁₋₈ alkyl, C₂₋₈    alkenyl, and C₁₋₈ haloalkyl;-   R³ is selected from the group consisting of —NR^(g)R^(h) and C₄₋₁₂    heterocyclyl, wherein the C₄₋₁₂ heterocyclyl is optionally    substituted with 1 to 6 R^(y);-   each R^(y) is independently selected from the group consisting of    -   halogen, —CN, —R^(i), —CO₂R^(j), —CONR^(j)R^(k), —CONHC₁₋₆        alkyl-OH, —C(O)R^(j), —OC(O)NR^(j)R^(k), —NR^(j)C(O)R^(k),        —NR^(j)C(O)₂R^(k), CONOH, PO₃H₂, —NR^(j)—C₁₋₆ alkyl-C(O)₂R^(k),        —NR^(j)C(O)NR^(j)R^(k), —NR^(j)R^(k), —OR^(j),        —S(O)₂NR^(j)R^(k), —O—C₁₋₆alkyl-OR^(j),        —O—C₁₋₆alkyl-NR^(j)R^(k), —O—C₁₋₆ alkyl-CO₂R^(j),        —O—C₁₋₆alkyl-CONR^(j)R^(k), —C₁₋₆ alkyl-OR^(j), —C₁₋₆        alkyl-NR^(j)R^(k), —C₁₋₆ alkyl-CO₂R^(j), —C₁₋₆        alkyl-CONR^(j)R^(k), and SF₅,-   wherein the C₁₋₆ alkyl portion of R^(y) is optionally further    substituted with OH, SO₂NH₂, CONH₂, CONOH, PO₃H₂, COO—C₁₋₈alkyl or    CO₂H, wherein each R^(j) and R^(k) is independently selected from    hydrogen, C₁₋₈ alkyl optionally substituted with 1 to 2 substituents    selected from OH, SO₂NH₂, CONH₂, CONOH, PO₃H₂, COO—C₁₋₈alkyl or    CO₂H, and C₁₋₈ haloalkyl optionally substituted with 1 to 2    substituents selected from OH, SO₂NH₂, CONH₂, CONOH, PO₃H₂.    COO—C₁₋₈alkyl or CO₂H, or when attached to the same nitrogen atom    R^(j) and R^(k) can be combined with the nitrogen atom to form a    five or six-membered ring having from 0 to 2 additional heteroatoms    as ring members selected from N, O or S, and optionally substituted    with oxo; each R¹ is independently selected from the group    consisting of —OH, C₁₋₈ alkyl, C₂₋₈ alkenyl, and C₁₋₈ haloalkyl each    of which may be optionally substituted with OH, SO₂NH₂, CONH₂,    CONOH, PO₃H₂, COO—C₁₋₈ alkyl or CO₂H;-   R^(g) is selected from the group consisting of H, C₁₋₈ haloalkyl and    C₁₋₈ alkyl;-   R^(h) is selected from —C₁₋₈ alkyl, C₁₋₈ haloalkyl, C₁₋₈ alkyl-COOH,    C₁₋₈ alkyl-OH, C₁₋₈ alkyl-CONH₂, C₁₋₈ alkyl-SO₂NH₂, C₁₋₈    alkyl-PO₃H₂, C₁₋₈ alkyl-CONOH, C₁₋₈ alkyl-NR^(h1)R^(h2),    —C(O)—C₁₋₈alkyl, —C(O)—C₁₋₈alkyl-OH, —C(O)—C₁₋₈alkyl-COOH, C₃₋₁₀    cycloalkyl, —C₃₋₁₀ cycloalkyl-COOH, —C₃₋₁₀ cycloalkyl-OH, C₄₋₈    heterocyclyl, —C₄₋₈ heterocyclyl-COOH, —C₄₋₈ heterocyclyl-OH, —C₁₋₈    alkyl-C₄₋₈ heterocyclyl, —C₁₋₈ alkyl-C₃₋₁₀ cycloalkyl, C₅₋₁₀    heteroaryl, —C₁₋₈alkyl-C₅₋₁₀ heteroaryl, C₁₀ carbocyclyl, —C₁₋₈    alkyl-C₆₋₁₀ aryl, —C₁₋₈ alkyl-(C═O)—C₆₋₁₀ aryl, —C₁₋₈    alkyl-NH(C═O)—C₁₋₈ alkenyl, —C₁₋₈ alkyl-NH(C═O)—C₁₋₈ alkyl, —C₁₋₈    alkyl-NH(C═O)—C₁₋₈ alkynyl, —C₁₋₈ alkyl-(C═O)—NH—C₁₋₈ alkyl-COOH,    and —C₁₋₈ alkyl-(C═O)—NH—C₁₋₈ alkyl-OH optionally substituted with    CO₂H; or    -   R^(h) combined with the N to which it is attached is a mono-,        di- or tri-peptide comprising 1-3 natural amino acids and 0-2        non-natural amino acids, wherein    -   the non-natural aminoacids have an alpha carbon substituent        selected from the group consisting of C₂₋₄ hydroxyalkyl, C₁₋₃        alkyl-guanidinyl, and C₁₋₈ alkyl-heteroaryl,    -   the alpha carbon of each natural or non-natural amino acids are        optionally further substituted with a methyl group, and    -   the terminal moiety of the mono-, di-, or tri-peptide is        selected from the group consisting of C(O)OH, C(O)O—C₁₋₆ alkyl,        and PO₃H₂, wherein    -   R^(h1) and R^(h2) are each independently selected from the group        consisting of H, C₁₋₆ alkyl, and C₁₋₄ hydroxyalkyl;    -   the C₁₋₈ alkyl portions of R^(h) are optionally further        substituted with from 1 to 3 substituents independently selected        from OH, COOH, SO₂NH₂, CONH₂, CONOH, COO—C₁₋₈ alkyl, PO₃H₂ and        C₅₋₆ heteroaryl optionally substituted with 1 to 2 C₁₋₃ alkyl        substituents,    -   the C₁₀ carbocyclyl, C₅₋₁₀ heteroaryl and the C₆₋₁₀ aryl        portions of R^(h) are optionally substituted with 1 to 3        substituents independently selected from OH, B(OH)₂, COOH,        SO₂NH₂, CONH₂, CONOH, PO₃H₂, COO—C₁₋₈alkyl, C₁₋₄alkyl,        C₁₋₄alkyl-OH, C₁₋₄alkyl-SO₂NH₂, C₁₋₄alkyl CONH₂,        C₁₋₄alkyl-CONOH, C₁₋₄alkyl-PO₃H₂, C₁₋₄alkyl-COOH, and phenyl and    -   the C₄₋₈ heterocyclyl and C₃₋₁₀ cycloalkyl portions of R^(h) are        optionally substituted with 1 to 4 R^(w) substituents;-   each R^(w) substituent is independently selected from C₁₋₄ alkyl,    C₁₋₄ alkyl-OH, C₁₋₄ alkyl-COOH, C₁₋₄ alkyl-SO₂NH₂, C₁₋₄ alkyl CONH₂,    C₁₋₄ alkyl-CONOH, C₁₋₄ alkyl-PO₃H, OH, COO—C₁₋₈ alkyl, COOH, SO₂NH₂,    CONH₂, CONOH, PO₃H₂ and oxo;-   R⁴ is selected from the group consisting of O—C₁₋₈ alkyl, O—C₁₋₈    haloalkyl, O—C₁₋₈ alkyl-R^(z), C₆₋₁₀ aryl, C₅₋₁₀ heteroaryl, —O—C₁₋₄    alkyl-C₆₋₁₀ aryl and —O—C₁₋₄ alkyl-C₅₋₁₀ heteroaryl, wherein the    C₆₋₁₀ aryl and the C₅₋₁₀ heteroaryl are optionally substituted with    1 to 5 R^(z);-   each R^(z) is independently selected from the group consisting of    halogen, —CN, —R^(m), —CO₂R^(n), —CONR^(n)R^(p), —C(O)R^(n),    —OC(O)NR^(n)R^(p), —NR^(n)C(O)R^(p), —NR^(n)C(O)₂R^(m),    —NR^(n)—C(O)NR^(n)R^(p), —NR^(n)R^(p), —OR^(n), —O—X³—OR^(n),    —O—X³—NR^(n)R^(p), —O—X³—CO₂R^(n), —O—X³—CONR^(n)R^(p), —X³—OR^(n),    —X³—NR^(n)R^(p), —X³—CO₂R^(n), —X³—CONR^(n)R^(p), —SF₅,    —S(O)₂R^(n)R^(p), —S(O)₂NR^(n)R^(p), and three to seven-membered    carbocyclic or four to seven-membered heterocyclic ring wherein the    three to seven-membered carbocyclic or four to seven-membered    heterocyclic ring is optionally substituted with 1 to 5 R^(t),    wherein each R^(t) is independently selected from the group    consisting of C₁₋₈ alkyl,    -   C₁₋₈haloalkyl, —CO₂R^(n), —CONR^(n)R^(p), —C(O)R^(n),    -   —OC(O)NR^(n)R^(p), —NR″C(O)R^(p), —NR^(n)C(O)₂R^(m),        —NR^(n)—C(O)NR^(n)R^(p), —NR^(n)R^(p), —OR^(n), —O—X³—OR^(n),        —O—X³—NR^(n)R^(p), —O—X³—CO₂R^(n), —O—X³—CONR^(n)R^(p),        —X³—OR^(n), —X³—NR^(n)R^(p), —X³—CO₂R^(n), —X³—CONR^(n)R^(p),        —SF₅, and —S(O)₂NR^(n)R^(p);    -   wherein each X³ is a C₁₋₄ alkylene; each R^(n) and R^(p) is        independently selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈        haloalkyl, or when attached to the same nitrogen atom can be        combined with the nitrogen atom to form a five or six-membered        ring having from 0 to 2 additional heteroatoms as ring members        selected from N, O or S, and optionally substituted with oxo;        each R^(m) is independently selected from the group consisting        of C₁₋₈ alkyl, C₂₋₈ alkenyl, and C₁₋₈ haloalkyl; and optionally        when two R^(z) substituents are on adjacent atoms, they are        combined to form a fused five or six-membered carbocyclic or        heterocyclic ring optionally substituted with oxo;-   n is 0, 1, 2 or 3;-   each R⁵ is independently selected from the group consisting of    halogen, —CN, —R^(q), —CO₂R^(r), —CONR^(r)R^(s), —C(O)R^(r),    —OC(O)NR^(r)R^(s), —NR^(r)C(O)R^(s), —NR^(r)C(O)₂R^(q),    —NR^(r)—C(O)NR^(r)R^(s), —NR^(r)R^(s), —OR^(r), —O—X⁴—OR^(r),    —O—X⁴—NR^(r)R^(s), —O—X⁴—CO₂R^(r), —O—X⁴—CONR^(r)R^(s), —X⁴—OR^(r),    —X⁴—NR^(r)R^(s), —X⁴—CO₂R^(r), —X⁴—CONR^(r)R^(s), —SF₅,    —S(O)₂NR^(r)R^(s), wherein each X⁴ is a C₁₋₄ alkylene; each R^(r)    and R^(s) is independently selected from hydrogen, C₁₋₈ alkyl, and    C₁₋₈ haloalkyl, or when attached to the same nitrogen atom can be    combined with the nitrogen atom to form a five or six-membered ring    having from 0 to 2 additional heteroatoms as ring members selected    from N, O or S, and optionally substituted with oxo; each R^(q) is    independently selected from the group consisting of C₁₋₈ alkyl, and    C₁₋₈ haloalkyl;-   R^(6a) is selected from the group consisting of H, C₁₋₄ alkyl and    C₁₋₄ haloalkyl;-   each R^(6b) is independently selected from the group consisting of    F, C₁₋₄ alkyl, O—R^(u), C₁₋₄ haloalkyl, NR^(u)R^(v), wherein each    R^(u) and R^(v) is independently selected from hydrogen, C₁₋₈ alkyl,    and C₁₋₈ haloalkyl, or when attached to the same nitrogen atom can    be combined with the nitrogen atom to form a five or six-membered    ring having from 0 to 2 additional heteroatoms as ring members    selected from N, O or S, and optionally substituted with oxo; and-   m is 0, 1, 2, 3 or 4.

In some embodiments, the small molecule PD-1/PD-L1 inhibitor is selectedfrom the compounds or pharmaceutical compositions disclosed in WO2018/005374 filed by ChemoCentryx on Jun. 26, 2017. The contents ofwhich is incorporated herein for all purposes.

The PD-1 and/or PD-L1 inhibitors of the present disclosure can beformulated to retard the degradation of the compound or antibody or tominimize the immunogenicity of the antibody. A variety of techniques areknown in the art to achieve this purposes.

In the combination therapy described herein, the CCR2 antagonist can beformulated together with the additional therapeutic agent or separately.Both the CCR2 antagonist and the additional therapy will be formulatedin suitable dosage unit formulations (either alone or together)containing conventional nontoxic pharmaceutically acceptable carriers,adjuvants and vehicles appropriate for each rouse of administration. Itwill be understood, that the specific dose level and frequency of dosagefor any particular patient may be varied and will depend upon a varietyof factors including the activity of the specific compound employed, themetabolic stability and length of action of that compound, the age, bodyweight, hereditary characteristics, general health, sex, diet, mode andtime of administration, rate of excretion, drug combination, theseverity of the particular condition, and the host undergoing therapy.

Biological products such as antibodies may be constituted in apharmaceutical composition containing one or antibodies or a fragmentthereof and a pharmaceutically acceptable carrier. As used herein, a“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Apharmaceutical composition of the invention may include one or morepharmaceutically acceptable salts, anti-oxidant, aqueous and nonaqueouscarriers, and/or adjuvants such as preservatives, wetting agents,emulsifying agents and dispersing agents.

In some embodiments, the therapeutic compound and agent are eachprovided in an amount that would be sub-therapeutic if provided alone orwithout the other. Those of skill in the art will appreciate that“combinations” can involve combinations in treatments (i.e., two or moredrugs can be administered as a mixture, or at least concurrently or atleast introduced into a subject at different times but such that bothare in a subject at the same time).

Likewise, compounds, agents and compositions of the present inventionmay be used in combination with other drugs that are used in thetreatment, prevention, suppression or amelioration of cancer. Such otherdrugs may be administered, by a route and in an amount commonly usedtherefor, contemporaneously or sequentially with a compound, agent orcomposition of the present invention. When a compound, agent orcomposition of the present invention is used contemporaneously with oneor more other drugs, a pharmaceutical composition containing such otherdrugs in addition to the compound, agent or composition of the presentinvention is preferred. Accordingly, pharmaceutical compositions caninclude those that also contain one or more other active ingredients ortherapeutic agents, in addition to a compound, agent or composition ofthe present invention.

Combination therapy includes co-administration of the CCR2 antagonistand an additional therapeutic agent, sequential administration of theCCR2 antagonist and an additional therapeutic agent, administration of acomposition containing the CCR2 antagonist and an additional therapeuticagent 1 inhibitor, or simultaneous administration of separatecompositions such that one composition contains the CCR2 antagonist andanother composition contains an additional therapeutic agent.

IV. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Materials & Methods

Animals and Cell Lines.

Female C57BL/6 mice (6-8 weeks old) were purchased from Charles River(Hollister, Calif.) and housed in animal facilities at University ofCalifornia Davis (UCDAVIS), Sacramento, Calif. All animal experimentswere conducted in accordance with the guidelines and approval of theInstitutional Animal Care and Usage Committee at UCDAVIS. The MBL2 cellline is an established cell line derived from Moloney MuLV-inducedT-cell lymphoma in C57BL/6 mice, in which the gag gene was deleted fromthe genome in order to lower virus-dependent immunogenicity. MBL2 cellswere cultured in DMEM (Invitrogen, Carlsbad, Calif.) with 100%heat-inactivated FBS.

Establishment of MBL2 Tumors in Mice.

The method of establishing mouse ear skin tumors was previouslydescribed [28]. Briefly, PBS-washed MBL2 cells (4×10⁵ in 20 μl PBS) wereinjected into the dermal space under the central dorsal surface of theears and above the cartilage plane using a 28 g needle. Mice were thentopically treated one time with DNFB (1-Fluoro-2, 4-dinitrobenzene, 0.5%in a vehicle consisting of 4:1 (v/v) acetone and olive oil, 10 μl/ear)(Sigma, St. Louis, Calif.) on dorsal ear skins. Tumor growth wasassessed for maximum ear thickness using a digital caliper, or by weightfor the whole tumor-bearing ears removed from ear baseline. The endpointdetermination is based on the allowed maximal ear tumor size, or localerosion and bleeding, which usually occurred within two weeks afterimplantation.

Tumor Treatment by CCR2 Antagonist and/or Anti-PD1.

Small molecule compounds, Compound 1, either in a high concentration (6mg/ml) or a low concentration (2 mg/ml), as well as vehicle control wereall provided by ChemoCentryx (Mountain View, Calif.) in a lab-readyformulation. In therapeutic application, oral administration throughgavage of Compound 1 or vehicle started on the same day of MBL2 cellinoculation, usually two hours apart. Compound 1 was administrated oncea day (60 mg/kg for high dose or 20 mg/kg for low dose) for up to twoweeks following tumor implantation. Mice were euthanized on day 3 or day7 for analysis of early immune responses to the treatment. For tumortreatment with anti-PD1, in vivo MAb anti-mouse PD-1 (CD279) andrat-IgG2a (BioXcell, West Lebanon, N.H.) were injected via IP (10 mg/kgper mouse) three times a week starting on the same day of tumorimplantation. For combination therapy, the above single agent regimenwas kept the same.

H&E and Histoimmunochemistry.

After mouse ear tumors were surgically removed at the ear base, the earsample was cut into two parts along the long axis and placed in RNAlaterfor RNA extraction or into 10% formalin for hematoxylin and eosin (H&E)staining or immunohistochemical staining with purified mouse antibodies(anti-CD8 and anti-F4/80 from Biolegend, San Diego, Calif.).

Quantitative Real-Time PCR.

RNA (<2 μg per sample) was converted into cDNA with the high-capacityfirst-strand cDNA Kit (Qiagen). Real-time PCR was performed on a StepOnePlus Real-time PCR system (Applied Biosystem, Carlsbad, Calif.). QPCRprimer paires were purchased from Integrated DNA technologies(Coralville, Iowa).

CD8 T Cell Depletion in Tumor Model.

InVivoPlus anti-mouse CD8α (Clone 53-6.7), purchased from BioXcell, wasinjected via intraperitoneal route (250 μg per injection) in mice theday before tumor implantation. A second injection was performed after 7days with the same dose. To analyze the effect of CD8 depletion, eartumor-inoculated mice were euthanized three days after the firstadministration. Cervical draining lymph nodes were collected and cellsuspension was isolated for flow cytometry analysis that includedstaining with FITC-anti-CD8 (a different clone 5H10-1, Biolegend, SanDiego, Calif.).

Flow Cytometry for Mouse Ear Tissues, Lymph Nodes, and Spleens.

Anti-mouse CD45 (clone 30-F11), CD11b (M1/70), F4/80 (BM8), Ly6G (1A8),Ly6C (HK1.4), IFN-γ (XMG1.2) and CD8 (5H10-1) Abs were purchased fromBioLegend (San Diego, Calif.). Ears or tumor tissues were digested toobtain skin cell suspensions as described [30]. Lymph nodes or spleenswere directly minced and filtered through cell strainers with 100 μmmicron pores (Thermo Fisher Scientific, Waltham, Mass.). Red blood cellsin the spleen samples were removed by RBC lysis buffer (BioLegend).Intracellular staining was done after incubating cells for 4 h withbrefeldin A and PMA/ionomycin as described. Flow cytometry was performedusing an Acuri C6 or LSR II (BD Biosciences, San Jose, Calif.) inconjunction with FlowJo analysis software (Tree Star, San Carlos,Calif.).

Statistical Analysis.

All data are expressed as mean±SEM. Data were analyzed using GraphPadPrism version 6 (GraphPad Software, San Diego, Calif.). Simplecomparisons of means and SEM of data were made by using a two-sidedStudent t test. A p value <0.05 was considered statisticallysignificant.

Example 1: A Small Molecule CCR2 Antagonist Depletes Tumor Macrophagesand Stimulates CD8 T Cell Accumulation in a Murine Model of Cutaneous TCell Lymphoma (CTCL) Summary

Tumor-associated macrophages (TAMs) recruited from blood monocytes havebeen implicated to play a critical role in establishing animmunosuppressive tumor microenvironment (TME) that supports tumorgrowth. We have reported the establishment of high grade skin T celllymphoma in syngeneic mouse skin by injection of MBL2 T lymphoma cellsin ear skin followed by application of DNFB. In this model, macrophagesplay a key role in sustaining tumor growth. Thus, we hypothesize thatblocking monocyte trafficking (through inhibition of specific chemokinereceptors) into skin can influence tumor development. Herein, we examinethe effects of oral administration of a small molecule drug, Compound 1,that blocks CCR2-mediated chemotaxis of monocytes in this tumor model.Following Compound 1 administration for two days after tumor initiation,we measured (by flow cytometry) a marked depletion of macrophages in theskin (17.7% of total leukocytes vs. 2.78% in vehicle- and Compound1-treated mice, respectively). One week after treatment, neutrophilicabscesses and epidermal ulceration occurred at the tumor site ofCompound 1-, but not vehicle-treated, mice. Flow cytometry identifiedsignificantly larger numbers of neutrophils in the TME followingCompound 1 treatment. At two weeks, most of the mice in control groupwere euthanized because of large tumors. However, Compound 1-treatedtumors were smaller and sometimes nearly eradicated because of anintense inflammatory response comprised of significantly larger numbersof CD8+ T cells within the tumor (identified by immunohistochemistry).In summary, our data show a marked reduction of tumoral macrophageaccumulation in Compound 1-treated mice accompanied in many animals by areduction in tumor size and an increase in CD8+ T cells in the TME. Wesuggest that a therapeutic strategy for CTCL based on inhibition of theCCR2 receptor and regulation of the tumor microenvironment warrantsfurther exploration.

Example 2: Compound 1, a CCR2 Antagonist, Inhibited Tumor Progression ina Mouse Model of Skin T Cell Lymphoma

We have previously reported an inflammation-dependent mouse T celllymphoma model that was generated by implantation of MBL2 cells insubcutaneous skin followed by a single topical application of 2,4-Dinitro-1-fluorobenzene (DNFB) in the ears. Implantation of MBL2 cellsalone in the subcutaneous ears, though in syngeneic mice, does notresult in tumor formation, presumably because the inflammation triggeredby DNFB is often required for efficient tumor formation. However, whenmice are applied a single dose of DNFB, a well-studied contact allergen,on the dorsal skin immediately following tumor cell implantation, theresultant tumor microenvironments (TME) allows reproducible tumorgeneration in two weeks. The application of DNFB induces large amount ofinflammatory cells infiltrating in the TME, which contains mainlymyeloid cell populations, i.e. macrophages and neutrophils. By inducingmacrophage “suicide” using clodronate liposomes, we have shown that themacrophages in the MBL2/DNFB model contribute to tumor growth [29].Therefore, we hypothesize that compounds targeting the chemokinereceptor CCR2 for blocking monocytes recruitment and macrophagedifferentiation in the TME would also potentially reduce growth of Tcell lymphoma tumors in the skin.

Compound 1 is an orally-bioavailable CCR2 antagonist. After beingadministered with two different doses, 20 mg/kg or 60 mg/kg, throughdaily oral gavage, plasma concentration of Compound 1 in mice correlatedwell to the feeding doses (FIG. 2A). In addition, neither of the dosingschemes resulted in significant (>20%) weight changes in the mice (FIG.2B), suggesting that the drug was well tolerated. For the experimentaltreatment regimen, we administered Compound 1 daily via oral gavage,starting on the same day of tumor implantation (FIG. 1a ). Ear tumors inthe mice treated with two different doses of Compound 1, but not thevehicle treated mice, showed visible reduction in tumor growth (FIG.1C). Both ear thickness and ear weight (measured immediately aftereuthanasia) were significantly reduced with Compound 1 treatmentcompared to either the untreated group or vehicle-treated control (FIG.1C). Thus, Compound 1 blocked tumor growth in an inflammation andmacrophages dependent model of T cell lymphoma.

Example 3: CCR2⁺ Macrophages are Specifically Depleted in the/TumorMicroenvironment (TME) after Compound 1 Treatment

As we have previously shown, DNFB induces an inflammatory TME in the earskin in the MBL2/DNFB model, i.e. ears exhibit redness, edema, and rapidaccumulation of large numbers of inflammatory cells in just two days[28]. In order to reveal the mechanisms by which Compound 1 reducestumor growth in mice, we examined mice that were treated with Compound 1for two days after tumor implantation. Flow cytometry analysis of cellsuspension from the ear tumors showed that levels of CD11b⁺/F4/80⁺macrophages were significantly decreased by the Compound 1 treatment,which included both percentage values of live cells and absolute numbersby calculation in whole ears (FIGS. 3A & B). Since both flow data andtumor measurement indicated that the higher dosage of Compound 1generated better treatment outcomes without side effects, we used 60mg/kg per day as a standard dosage in subsequent experiments.

It is known that other myeloid-derived subpopulations that are closelyrelated to macrophages in terms of immune function accumulate in theTME. We wondered if Compound 1 specifically targets the CD11b and F4/80positive macrophages detected above. Combining the surface markers, i.e.CD11b, F4/80, Ly6G, Ly6C and CCR2, by flow cytometry on single cellsuspension from the whole ear tissues, we saw that there were clearlytwo types of cells that dominated the myeloid cell population gated onCD11b (FIG. 3C). F4/80 positive cells, however, are the cell type thatis targeted by Compound 1. This population co-expresses Ly6C and CCR2,the functional target of Compound 1. The other major cell population iscomprised of CD11b- and Ly6G-positive cells, which also co-express Ly6C,but not CCR2 (FIG. 3C). Although these cells show neutrophil markers, wecall them neutrophil-like cells because of their potentially immaturenature and features similar to MDSCs (myeloid-derived suppressor cells)in the setting of the tumor microenvironment. As shown in FIG. 3C,accumulation of neutrophils was not blocked by Compound 1; on thecontrary, their relative abundance increased because of the depletion ofmacrophages by CCR2 antagonism.

Example 4: Compound 1 Treatment Enhances Intratumoral Inflammation

During tumor formation, the mice treated with Compound 1 showedsignificantly enhanced skin inflammation in the ears, which were redderand scalier than the control mice. Histological examination of tissuesfrom day 7 revealed that ear surfaces on the dorsal side, i.e.DNFB-exposed side, exhibited surface ulceration, scaling, and obviousaccumulation of inflammatory infiltrates microscopically (FIG. 4A). IHCstaining with anti-F4/80 confirmed that macrophages were largely absentin the TME (FIG. 4B). Flow cytometry analysis on the tissues from thesame time point showed a significant increase of neutrophil-like cells,which is consistent with histological manifestation (FIG. 4C). Of note,not only did the percentage increase, but also the total numbersincreased, indicating that neutrophil-like cells were recruited to theTME accompanying the macrophage depletion. Thus, treatment with Compound1 results in tumor cell necrosis and an increase in neutrophil-likecells in a TME that possesses low numbers of F4/80+ macrophages.

Example 5: Compound 1 Treatment Alters Cytokine and Immune Biomarkers inthe TME

To further understand mechanisms underlying Compound 1-mediated tumorinhibition, we quantified cytokines and chemokines known to be involvedin anti-tumor immunity in Compound 1-treated tumors. Of interest, IFN-γ,IFN-γ-induced chemokines, CXCL10 and CXCL11 were all significantlyincreased in ears at the mRNA level by treatment with Compound 1. IL-12,another Th1 marker, was found to increase in Compound 1-treated mouseears. We saw a consistent upregulation of granzyme B, another indicationof activation of anti-tumor cytotoxic pathways (FIG. 5A). By contrast,expression of IL-10 and TGF-beta, representative Th2 cytokines, weresimilarly expressed between Compound 1-treated mice and controls (FIG.6).

Additional analysis of gene expression showed that several majorinflammatory cytokines, such as IL-17a, IL-1beta, and IL-6 wereupregulated to variable extents in Compound 1-treated mice. Upregulationof CCL2, the ligand of CCR2, and its closely related chemokine CCL7,during CCR2 antagonism in the TME may reflect enhanced transcription ofCCL2 in the setting of effective CCR2 inhibition (FIG. 5B). The lastgroup of biomarkers noticeably increased in the TME after Compound 1treatment are recognized neutrophil chemoattractants and biomakers, i.e.CXCL1/2 and S100A8/9 (FIG. 5C), which is consistent with the recruitmentof neutrophil-like cells as shown by flow cytometry (FIG. 4C).

Example 6: CD8 T Cells Mediate the Anti-Tumor Activities FollowingMacrophage Blockade in the TME

We next asked if CD8 T cells were required for Compound 1 to effectivelyblock tumor growth. The tumor tissues were collected from tumor bearingmice receiving two weeks of treatment. Few CD8 T cells were observed inuntreated and vehicle treated tumors by IHC staining (FIG. 7A). Compound1 treatment, however, markedly increased the numbers of CD8⁺ T cellsinfiltration in the TME in a dose-dependent manner (FIG. 7A).

Next, we administrated neutralizing CD8 antibodies by IP injectionconcurrently with the Compound 1 treatment (FIG. 7B). Three days afterthe first injection of depleting anti-CD8 antibodies, we saw thatCD3+/CD8+ T cells were virtually absent in the cervical draining lymphnodes in the antibody-treated mice (FIG. 7C). Two weeks after anti-CD8treatment, measurement of the ear tumor size at the endpoint showedagain that Compound 1-treatment inhibited tumor growth as indicated(outlined green triangles), whereas anti-CD8 abrogated this effect(similar to the levels of the vehicle treated groups) (FIG. 7D). In theDNFB-MBL2 model, the size of the ipsilateral cervical draining lymphnodes correlated well with nodal metastasis. As shown in FIG. 7E,reduction of cervical LN size with Compound 1 treatment was reversed byCD8 T cells depletion. Thus, Compound 1 treatment requires the presenceof CD8+ T cells for effective reduction of tumor growth as well as LNmetastasis.

Example 7: Macrophage Blockade Synergizes with Anti-PD1 in ConstrainingMBL2 Tumor Growth

The role of PD1 in cancer immune evasion is well established in so faras tumor cells or antigen-presenting cells, such as macrophages, expressPD-L1 and interact with PD-1 positive CD8-T cells to render them anergicwith respect to antitumor activity. Thus, inhibitors blocking theinteraction between PD-1 and PD-L1 can enhance T-cell responses, knownas immune checkpoint blockade. Compared to the cultured MBL2 cells invitro, MBL2 tumor formed in mice exhibited a significant increase ofPD-L1 (FIG. 8A). Similar to results with Compound 1, antibody blockadeof PD-1/PD-L1 axis in this MBL2 model could effectively delay the tumorgrowth, but was unable to eliminate the tumors (FIG. 9A-B). Thisprompted us to apply combination therapy over a single agent in order toachieve better anti-tumor effects.

For the combination therapy, mice were treated with anti-PD1 at 10 mg/kgevery other day beginning on the same day of the first Compound 1treatment and tumor implantation (FIG. 8B). When mice were euthanizedafter completing the two weeks treatment, we analyzed the spleens ineach group and found that the number of IFN-γ-producing CD8-T cellssignificantly increased in the group treated with the combination ofCompound 1 and anti-PD1, suggesting these mice exhibit more robustanti-tumor immunity (FIG. 8C). In examining the size of ear tumors, wefound that the combination therapy, but not the anti-PD1 and vehicletreatment, significantly inhibited the ear tumor growth compared to thecontrol group treated with isotype and vehicle. According to our pastobservations of the MBL2/DNFB model for extended time periods, i.e. over6 weeks after tumor implantation, mice with an ear thickness less than 1mm at two weeks rarely, if ever, develop tumors. As shown by thehorizontal dotted line in the graph, nearly 75% of the mice fromcombined treatment group had a tumor thickness less than 1 mm,suggestive of long term tumor clearance (FIG. 8D).

REFERENCES

-   1. Grivennikov S I, Greten F R, Karin M: Immunity, inflammation, and    cancer. Cell 2010, 140:883-899.-   2. Franklin R A, Liao W, Sarkar A, Kim M V, Bivona M R, Liu K, Pamer    E G, Li M O: The cellular and molecular origin of tumor-associated    macrophages. Science 2014, 344:921-925.-   3. Yang L, Zhang Y: Tumor-associated macrophages: from basic    research to clinical application. J Hematol Oncol 2017, 10:58.-   4. Fujimura T, Kambayashi Y, Fujisawa Y, Hidaka T, Aiba S:    Tumor-Associated Macrophages: Therapeutic Targets for Skin Cancer.    Front Oncol 2018, 8:3.-   5. Fujimura T, Ring S, Umansky V, Mahnke K, Enk A H: Regulatory T    cells stimulate B7-H1 expression in myeloid-derived suppressor cells    in ret melanomas. J Invest Dermatol 2012, 132:1239-1246.-   6. Pedersen M B, Danielsen A V, Hamilton-Dutoit S J, Bendix K,    Norgaard P, Moiler M B, Steiniche T, d'Amore F: High intratumoral    macrophage content is an adverse prognostic feature in anaplastic    large cell lymphoma. Histopothology 2014, 65:490-500.-   7. Tang X, Mo C, Wang Y, Wei D, Xiao H: Anti-tumour strategies    aiming to target tumour-associated macrophages. Immunology 2013,    138:93-104.-   8. Cheung K J, Johnson N A, Affleck J G, Severson T, Steidl C,    Ben-Neriah S, Schein J, Morin R D, Moore R, Shah S P, et al:    Acquired TNFRSF14 mutations in follicular lymphoma are associated    with worse prognosis. Cancer Res 2010, 70:9166-9174.-   9. Stanley E R, Chitu V: CSF-1 receptor signaling in myeloid cells.    Cold Spring Harb Perspect Biol 2014, 6.-   10. Papadopoulos K P, Gluck L, Martin L P, Olszanski A J, Tolcher A    W, Ngarmchamnanrith G, Rasmussen E, Amore B M, Nagorsen D, Hill J S,    Stephenson J, Jr.: First-in-Human Study of AMG 820, a Monoclonal    Anti-Colony-Stimulating Factor 1 Receptor Antibody, in Patients with    Advanced Solid Tumors. Clin Cancer Res 2017, 23:5703-5710.-   11. Cannarile M A, Weisser M, Jacob W, Jegg A M, Ries C H, Ruttinger    D: Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancer    therapy. J Immunother Cancer 2017, 5:53.-   12. Quail D F, Bowman R L, Akkari L, Quick M L, Schuhmacher A J,    Huse J T, Holland E C, Sutton J C, Joyce J A: The tumor    microenvironment underlies acquired resistance to CSF-1R inhibition    in gliomas. Science 2016, 352:aad3018.-   13. Zhang J, Patel L, Pienta K J: Targeting chemokine (C-C motif)    ligand 2 (CCL2) as an example of translation of cancer molecular    biology to the clinic. Prog Mol Biol Transl Sci 2010, 95:31-53.-   14. Chen X, Wang Y, Nelson D, Tian S, Mulvey E, Patel B, Conti I,    Jaen J, Rollins B J: CCL2/CCR2 Regulates the Tumor Microenvironment    in HER-2/neu-Driven Mammary Carcinomas in Mice. PLoS One 2016,    11:e0165595.-   15. Yao M, Smart C, Hu Q, Cheng N: Continuous Delivery of    Neutralizing Antibodies Elevate CCL2 Levels in Mice Bearing    MCF10CA1d Breast Tumor Xenografts. Transl Oncol 2017, 10:734-743.-   16. Pienta K J, Machiels J P, Schrijvers D, Alekseev B, Shkolnik M,    Crabb S J, Li S, Seetharam S, Puchalski T A, Takimoto C, et al:    Phase 2 study of carlumab (CNTO 888), a human monoclonal antibody    against CC-chemokine ligand 2 (CCL2), in metastatic    castration-resistant prostate cancer. Invest New Drugs 2013,    31:760-768.-   17. Sandhu S K, Papadopoulos K, Fong P C, Patnaik A, Messiou C,    Olmos D, Wang G, Tromp B J, Puchalski T A, Balkwill F, et al: A    first-in-human, first-in-class, phase I study of carlumab (CNTO    888), a human monoclonal antibody against CC-chemokine ligand 2 in    patients with solid tumors. Cancer Chemother Pharmacol 2013,    71:1041-1050.-   18. Lim S Y, Yuzhalin A E, Gordon-Weeks A N, Muschel R J: Targeting    the CCL2-CCR2 signaling axis in cancer metastasis. Oncotarget 2016,    7:28697-28710.-   19. Yao W, Ba Q, Li X, Li H, Zhang S, Yuan Y, Wang F, Duan X, Li J,    Zhang W, Wang H: A Natural CCR2 Antagonist Relieves Tumor-associated    Macrophage-mediated Immunosuppression to Produce a Therapeutic    Effect for Liver Cancer. E Bio Medicine 2017, 22:58-67.-   20. Zheng Y, Qin L, Zacarias N V, de Vries H, Han G W, Gustavsson M,    Dabros M, Zhao C, Cherney R J, Carter P, et al: Structure of CC    chemokine receptor 2 with orthosteric and allosteric antagonists.    Nature 2016, 540:458-461.-   21. Nywening T M, Wang-Gillam A, Sanford D E, Belt B A, Panni R Z,    Cusworth B M, Toriola A T, Nieman R K, Worley L A, Yano M, et al:    Targeting tumour-associated macrophages with CCR2 inhibition in    combination with FOLFIRINOX in patients with borderline resectable    and locally advanced pancreatic cancer: a single-centre, open-label,    dose-finding, non-randomised, phase 1b trial. Lancet Oncol 2016,    17:651-662.-   22. Roblek M, Calin M, Schlesinger M, Stan D, Zeisig R, Simionescu    M, Bendas G, Borsig L: Targeted delivery of CCR2 antagonist to    activated pulmonary endothelium prevents metastasis. J Control    Release 2015, 220:341-347.-   23. Hwang S T, Janik J E, Jaffe E S, Wilson W H: Mycosis fungoides    and Sezary syndrome. Lancet 2008, 371:945-957.-   24. Krejsgaard T, Lindahl L M, Mongan N P, Wasik M A, Litvinov I V,    Iversen L, Langhoff E, Woetmann A, Odum N: Malignant inflammation in    cutaneous T-cell lymphoma-a hostile takeover. Semin Immunopathol    2017, 39:269-282.-   25. Wu X, Hwang S T: Cutaneous T-Cell Lymphoma: The Yin and Yang of    Inflammation and Neoplasia. J Investig Dermatol Symp Proc 2015,    17:34-35.-   26. Sugaya M, Miyagaki T, Ohmatsu H, Suga H, Kai H, Kamata M, Fujita    H, Asano Y, Tada Y, Kadono T, et al: Association of the numbers of    CD163(+) cells in lesional skin and serum levels of soluble CD163    with disease progression of cutaneous T cell lymphoma. J Dermatol    Sci 2012, 68:45-51.-   27. Miyagaki T, Sugaya M, Suga H, Ohmatsu H, Fujita H, Asano Y, Tada    Y, Kadono T, Sato 5: Increased CCL18 expression in patients with    cutaneous T-cell lymphoma: association with disease severity and    prognosis. J Eur Acad Dermatol Venereal 2013, 27:e60-67.-   28. Wu X, Sells R E, Hwang S T: Upregulation of inflammatory    cytokines and oncogenic signal pathways preceding tumor formation in    a murine model of T-cell lymphoma in skin. J Invest Dermotol 2011,    131:1727-1734.-   29. Wu X, Schulte B C, Zhou Y, Haribhai D, Mackinnon A C, Plaza J A,    Williams C B, Hwang S T: Depletion of M2-like tumor-associated    macrophages delays cutaneous T-cell lymphoma development in vivo. J    Invest Dermotol 2014, 134:2814-2822.-   30. Broggi A, Cigni C, Zanoni I, Granucci F: Preparation of    Single-cell Suspensions for Cytofluorimetric Analysis from Different    Mouse Skin Regions. J Vis Exp 2016:e52589.-   31. Bonapace L, Coissieux M M, Wyckoff J, Mertz K D, Varga Z, Junt    T, Bentires-Alj M: Cessation of CCL2 Inhibition accelerates breast    cancer metastasis by promoting anglogenesis. Nature 2014,    515:130-133.-   32. Hitchcock J R, Watson C J: Anti-CCL2: building a reservoir or    opening the floodgates to metastasis? Breast Cancer Res 2015, 17:68.-   33. Xue C B, Wang A, Han Q, Zhang Y, Cao G, Feng H, Huang T, Zheng    C, Xia M, Zhang K, et al: Discovery of INCB8761/PF-4136309, a    Potent, Selective, and Orally Bioavailable CCR2 Antagonist. ACS Med    Chem Lett 2011, 2:913-918.-   34. Wein L, Savas P, Luen S J, Virassamy B, Salgado R, Loi S:    Clinical Validity and Utility of Tumor-Infiltrating Lymphocytes in    Routine Clinical Practice for Breast Cancer Patients: Current and    Future Directions. Front Oncol 2017, 7:156.-   35. Cohen I I, Blasberg R: Impact of the Tumor Microenvironment on    Tumor-Infiltrating Lymphocytes: Focus on Breast Cancer. Breast    Cancer (Auckl) 2017, 11:1178223417731565.-   36. Peranzoni E, Lemoine J, Vimeux L, Feuillet V, Barrin S,    Kantari-Mimoun C, Bercovici N, Guerin M, Biton J, Ouakrim H, et al:    Macrophages impede CD8 T cells from reaching tumor cells and limit    the efficacy of anti-PD-1 treatment. Proc Natl Acad Sci USA 2018,    115:E4041-E4050.-   37. Ahmadzadeh M, Johnson L A, Heemskerk B, Wunderlich J R, Dudley M    E, White D E, Rosenberg S A: Tumor antigen-specific CD8 T cells    infiltrating the tumor express high levels of PD-1 and are    functionally impaired. Blood 2009, 114:1537-1544.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

What is claimed is:
 1. A method of treating cutaneous T cell lymphoma(CTCL), said method comprising administering to a subject in needthereof an effective amount of a Chemokine Receptor 2 (CCR2) antagonist,wherein said CCR2 antagonist is a compound of Formula (Ia4)

or a pharmaceutically acceptable salt thereof; wherein the subscripts mand n are each independently 1; R³ is selected from the group consistingof C₁₋₈ alkyl and C₃₋₈ cycloalkyl; R⁴ is selected from the groupconsisting of H, and C₁₋₈ alkyl; the subscript p is 1 or 2; each R^(x)is independently selected from the group consisting of halogen, —R^(c),—OR^(a), —O—X¹—OR^(a), and —X¹—OR^(a), wherein each X¹ is a C₁₋₄alkylene; each R^(a) is selected from hydrogen, C₁₋₈ alkyl, and C₁₋₈haloalkyl; each R^(c) is independently selected from the groupconsisting of C₁₋₈ alkyl and C₁₋₈ haloalkyl; the subscript q is 1 or 2;each R^(z) is independently selected from the group consisting ofhalogen, R^(i), —CO₂R^(g), and tetrazole; wherein each R^(g) is selectedfrom hydrogen, C₁₋₈ alkyl, and C₁₋₈ haloalkyl; each R^(i) isindependently selected from the group consisting of C₁₋₈ alkyl, and C₁₋₈haloalkyl.
 2. The method of claim 1, wherein the subject in need thereofhas Stage IA CTCL.
 3. The method of claim 1, wherein the subject in needthereof has Stage IB CTCL.
 4. The method of claim 1, wherein the subjectin need thereof has Stage IIA CTCL.
 5. The method of claim 1, whereinthe subject in need thereof has Stage IIB CTCL.
 6. The method of claim1, wherein the subject in need thereof has Stage III CTCL.
 7. The methodof claim 1, wherein the subject in need thereof has Stage IV CTCL. 8.The method of claim 1, wherein said CCR2 antagonist is selected from thegroup consisting of

or a pharmaceutically acceptable salt thereof.
 9. The method of claim 1,wherein said CCR2 antagonist has the formula of Compound 1

or a pharmaceutically acceptable salt thereof.
 10. The method of claim1, wherein said CCR2 antagonist has the formula of Compound 2

or a pharmaceutically acceptable salt thereof.
 11. The method of claim1, further comprising administering one or more additional therapeuticagents.
 12. The method of claim 11, wherein the one or more additionaltherapeutic agents is an immune-checkpoint inhibitor.
 13. The method ofclaim 12, wherein the immune-checkpoint inhibitor is a PD-1 and/or PD-L1inhibitor.
 14. The method of claim 13, wherein the PD-1 and/or PD-L1inhibitor is selected from the group consisting of pembrolizumab,nivolumab, IBI-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591,PDR-001, GB-226, and STI-1110.
 15. The method of claim 13, wherein thePD-1 and/or PD-L1 inhibitor is selected from the group consisting ofNivolumab, Pembrolizumab, and Pidilizumab.
 16. The method of claim 13,wherein the PD-1 and/or PD-L1 inhibitor is selected from the groupconsisting of durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL,TSR-042, KD-033, CA-170, CA-327, STI-1014, and KY-1003.